Isolated nucleic acid sequence comprising SEQ ID NO: 14 and methods of using said nucleic acid sequence

ABSTRACT

The present invention provides methods of using Zven1 and Zven2 polypeptides to increase chemokine production. The present invention also provides methods for treating intestinal motility disorders and improving gastrointestinal function with Zven1 and Zven2 polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/680,800, filed Oct. 7, 2003 is now U.S. Pat. No. 7,291,589, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/416,719,filed Oct. 7, 2002; U.S. Provisional Application Ser. No. 60/416,718,filed Oct. 7, 2002; U.S. Provisional Application Ser. No. 60/434,116,filed Dec. 16, 2002; U.S. Provisional Application Ser. No. 60/433,918,filed Dec. 16, 2002; U.S. Provisional Application Ser. No. 60/508,614,filed Oct. 3, 2003; and U.S. Provisional Application Ser. No.60/508,603, filed Oct. 3, 2003, all of which are herein incorporated byreference.

BACKGROUND OF THE INVENTION

Optimal gastrointestinal function includes mixing and forward propulsionof contents in the stomach and intestine. Gastric emptying is frequentlyabnormal in patients with critical illness or who are recovering fromsurgery. Recovery of gastrointestinal function and resumption of oralintake are important determinants in recovery from an event thatcompromises gastrointestinal function. Several events can lead todysfunction in the gastrointestinal system, including, for example,ileus (post-operative and paralytic), chronic constipation,gastroparesis (including diabetic gastroparesis), intestinalpseudo-obstruction, dyspepsia, gastroesophageal reflux, and emesis.

Diseases and disorders of impaired or compromised gastrointestinalfunction include ileus and gastroparesis. Post-operative ileus (POI) isa condition of reduced intestinal tract motility, including delayedgastric emptying, that occurs as a result of disrupted muscle tonefollowing surgery. It is especially problematic following abdominalsurgery. The problem may arise from the surgery itself, from theresidual effects of anesthetic agents, and particularly, frompain-relieving narcotic and opiate drugs used during and after surgery.Post-operative ileus can be categorized as “uncomplicated”, lasting twoto three days after surgery, or as “paralytic”, lasting more than threedays after surgery. Thus, patients undergoing abdominal surgery who havea delay in recovery of gastrointestinal function have prolonged hospitalstays, which can lead to increased medical costs and potentially toother complications. An estimated 750 million to one billion dollars isspent each year in increased hospitalization due to post-operativeileus. Currently there are no drugs that have been approved fortreatment of this disease.

In addition to the need for a better therapeutic for post-operativeileus, there is a need for a better therapeutic for diabeticgastroparesis. Diabetic gastroparesis is paralysis of the stomachbrought about by a motor abnormality in the stomach, as a complicationof both type I and type II diabetes. Diabetic gastroparesis ischaracterized by delayed gastric emptying, post-prandial distention,nausea and vomiting. In diabetes, it is thought to be due to aneuropathy, though it is also associated with loss of interstitial cellsof Cajal (ICC), which are the “pacemaker cells” of the gut.

In the U.S. alone, there are at least 16 million individuals withdiabetes, affecting approximately 7% of the population. The prevalenceis continuing to increase and is growing worldwide. Since up totwo-thirds of individuals with diabetes suffer from some degree ofgastroparesis, this problem is significant. Episodes are often acute,though long-term treatment is often required. Moreover, symptomsassociated with diabetic gastroparesis, such as delayed gastricemptying, and emesis can cause water and electrolyte imbalances, poorglycemic control, and ensuing complications. If severe enough, it mayrequire hospitalization for control of diabetes, and treatment withintravenous fluids and nutrition.

The often-acute nature of the episodes provides an opportunity to treatwith a prokinetic. Currently there are very few drugs that caneffectively treat diabetic gastroparesis, and those that are availablehave side effects and/or cannot be taken with other medications. Oraldrugs may not be tolerated during severe episodes, and thus, wouldrequire intravenous administration of a prokinetic. In the UnitedStates, only two agents, erythromycin and metoclopramide, are availableto treat gastroparesis.

Thus, a need still exists for therapeutic approaches to treatment ofgastric function disorders.

BRIEF SUMMARY OF THE INVENTION

The present invention provides proteins useful for the treatment inrecovery of gastronintestinal function and gastric emptying. Other usesof Zven1 and Zven2 polypeptides are described in more detail below.

DESCRIPTION OF THE INVENTION 1. Overview

The present invention is directed to novel uses of previously describedproteins, Zven1 and Zven2. See U.S. patent application Ser. No.09/712,529, now issued as U.S. Pat. No. 6,485,938. Zven1 and Zven2 arealso known in the industry as Prokineticin2 and Prokineticin1,respectively. As discussed herein, Zven1 and Zven2, as well as variantsand fragments thereof, can be used to regulate gastrointestinal functionand gastric emptying. Receptors for Zven1 (Prokineticin2) and Zven2(Prokineticin1) have been identified as G protein-coupled receptors,GPCR73a and GPCR73b. See Lin, D. et al., J. Biol. Chem. 277:19276-19280, 2002. The GPCR73a and GPCR73b receptors are also known asPK-R1 and PK-R2.

The present invention provides methods of using human Zven polypeptidesand nucleic acid molecules that encode human Zven polypeptides. Anillustrative nucleic acid molecule containing a sequence that encodesthe Zven1 polypeptide has the nucleotide sequence of SEQ ID NO:1. Theencoded polypeptide has the following amino acid sequence: MRSLCCAPLLLLLLLPPLLL TPRAGDAAVI TGACDKDSQC GGGMCCAVSI WVKSIRICTP MGKLGDSCHPLTRKVPFFGR RMHHTCPCLP GLACLRTSFN RFICLAQK (SEQ ID NO:2). Thus, the Zven1nucleotide sequence described herein encodes a polypeptide of 108 aminoacids. The putative signal sequences of Zven1 polypeptide reside atamino acid residues 1 to 20, 1 to 21, and 1 to 22 of SEQ ID NO:2. Themature form of the polypeptide comprises the amino acid sequence fromamino acid 28 to 108 as shown in SEQ ID NO:2.

A longer form of the sequence as shown in SEQ ID NO:2 is included in theinvention described herein. The longer form has the following amino acidsequence: MRSLCCAPLL LLLLLPPLLL TPRAGDAAVI TGACDKDSQC GGGMCCAVSIWVKSIRICTP MGKLGDSCHP LTRKNNFGNG RQERRKRKRS KRKKEVPFFG RRMHHTCPCLPGLACLRTSF NRFICLAQK (SEQ ID NO:29). The putative signal sequence of thelonger form has a mature form that comprises the amino acid sequencefrom amino acid 28 to 129 as shown in SEQ ID NO:29.

An illustrative nucleic acid molecule containing a sequence that encodesthe Zven2 polypeptide has the nucleotide sequence of SEQ ID NO:4. Theencoded polypeptide has the following amino acid sequence: MRGATRVSIMLLLVTVSDCA VITGACERDV QCGAGTCCAI SLWLRGLRMC TPLGREGEEC HPGSHKVPFFRKRKHHTCPC LPNLLCSRFP DGRYRCSMDL KNINF (SEQ ID NO:5). Thus, the Zven2nucleotide sequence described herein encodes a polypeptide of 105 aminoacids. The putative signal sequences of Zven2 polypeptide reside atamino acid residues 1 to 17, and 1 to 19 of SEQ ID NO:5.

As described below, the present invention provides isolated polypeptidescomprising an amino acid sequence that is at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% identical toamino acid residues 23 to 108 of SEQ ID NO:2, to amino acid residues 28to 108 of SEQ ID NO:2, or to amino acid residues 28 to 129 if SEQ IDNO:29. Certain of such isolated polypeptides can specifically bind withan antibody that specifically binds with a polypeptide consisting of theamino acid sequence of SEQ ID NO:2. Particular polypeptides can increaseor decrease gastric contractility, gastric emptying and/or intestinaltransit. An illustrative polypeptide is a polypeptide that comprises theamino acid sequence of SEQ ID NO:2.

Similarly, the present invention includes provides isolated polypeptidescomprising an amino acid sequence that is at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95% identical toamino acid residues 20 to 105 of SEQ ID NO:5, wherein such isolatedpolypeptides can specifically bind with an antibody that specificallybinds with a polypeptide consisting of the amino acid sequence of SEQ IDNO:5. An illustrative polypeptide is a polypeptide that comprises theamino acid sequence of SEQ ID NO:5.

The present invention also provides polypeptides comprising an aminoacid sequence selected from the group consisting of: (1) amino acidresidues 21 to 108 of SEQ ID NO:2, (2) amino acid residues 22 to 108 ofSEQ ID NO:2, (3) amino acid residues 23 to 108 of SEQ ID NO:2, (4) aminoacid residues 82 to 108 of SEQ ID NO:2, (5) amino acid residues 1 to 78(amide) of SEQ ID NO:2, (6) amino acid residues 1 to 79 of SEQ ID NO:2,(7) amino acid residues 21 to 78 (amide) of SEQ ID NO:2, (8) amino acidresidues 21 to 79 of SEQ ID NO:2, (9) amino acid residues 22 to 78(amide) of SEQ ID NO:2, (10) amino acid residues 22 to 79 of SEQ IDNO:2, (11) amino acid residues 23 to 78 (amide) of SEQ ID NO:2, (12)amino acid residues 23 to 79 of SEQ ID NO:2, (13) amino acid residues 20to 108 of SEQ ID NO:2, (14) amino acid residues 20 to 72 of SEQ ID NO:2,(15) amino acid residues 20 to 79 of SEQ ID NO:2, (16) amino acidresidues 20 to 79 (amide) of SEQ ID NO:2, (17) amino acid residues 21 to72 of SEQ ID NO:2, (18) amino acid residues 21 to 79 (amide) of SEQ IDNO:2, (19) amino acid residues 22 to 72 of SEQ ID NO:2, (20) amino acidresidues 22 to 79 (amide) of SEQ ID NO:2, (21) amino acid residues 23 to72 of SEQ ID NO:2, (22) amino acid residues 23 to 79 (amide) of SEQ IDNO:2, (23) amino acid residues 28 to 108 of SEQ ID NO:2, (24) amino acidresidues 28 to 72 of SEQ ID NO:2, (25) amino acid residues 28 to 79 ofSEQ ID NO:2, (26) amino acid residues 28 to 79 (amide) of SEQ ID NO:2,(27) amino acid residues 75 to 108 of SEQ ID NO:2, (28) amino acidresidues 75 to 79 of SEQ ID NO:2, and (29) amino acid residues 75 to 78(amide) of SEQ ID NO:2. Illustrative polypeptides consist of amino acidsequences (1) to (29). The present invention also included polypeptidecomprising an amino acid sequence comprising amino acid 28 to 129 asshown in SEQ ID NO:29, and/or fragments thereof.

The present invention further includes polypeptides comprising an aminoacid sequence selected from the group consisting of: (a) amino acidresidues 20 to 105 of SEQ ID NO:5, (b) amino acid residues 18 to 105 ofSEQ ID NO:5, (c) amino acid residues 1 to 70 of SEQ ID NO:5, (d) aminoacid residues 20 to 70 of SEQ ID NO:5, (e) amino acid residues 18 to 70of SEQ ID NO:5, (f) amino acid residues 76 to 105 of SEQ ID NO:5, (g)amino acid residues 66 to 105 of SEQ ID NO:5, and (h) amino acidresidues 82 to 105 of SEQ ID NO:5. Illustrative polypeptides consist ofamino acid sequences (a) to (h).

The present invention further provides antibodies and antibody fragmentsthat specifically bind with such polypeptides. Exemplary antibodiesinclude polyclonal antibodies, murine monoclonal antibodies, humanizedantibodies derived from murine monoclonal antibodies, and humanmonoclonal antibodies. Illustrative antibody fragments include F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv, and minimal recognition units. The presentinvention also includes anti-idiotype antibodies that specifically bindwith such antibodies or antibody fragments. The present inventionfurther includes compositions comprising a carrier and a peptide,polypeptide, antibody, or anti-idiotype antibody described herein.

The present invention also provides isolated nucleic acid molecules thatencode a Zven polypeptide, wherein the nucleic acid molecule is selectedfrom the group consisting of (a) a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:3, (b) a nucleic acid molecule encodingthe amino acid sequence of SEQ ID NO:2, (c) a nucleic acid molecule thatremains hybridized following stringent wash conditions to a nucleic acidmolecule consisting of the nucleotide sequence of nucleotides 66 to 161of SEQ ID NO:1, the nucleotide sequence of nucleotides 288 to 389 of SEQID NO:1, or to the complement of the nucleotide sequence of eithernucleotides 66 to 161 of SEQ ID NO:1 or nucleotides 288 to 389 of SEQ IDNO:1, (d) a nucleic acid molecule comprising the nucleotide sequence ofSEQ ID NO:6, (e) a nucleic acid molecule encoding the amino acidsequence of SEQ ID NO:5, (f) a nucleic acid molecule that remainshybridized following stringent wash conditions to a nucleic acidmolecule consisting of the nucleotide sequence of nucleotides 334 to 405of SEQ ID NO:4, or to the complement of the nucleotide sequence ofnucleotides 334 to 405 of SEQ ID NO:4.

Illustrative nucleic acid molecules include those in which anydifference between the amino acid sequence encoded by the nucleic acidmolecule and the corresponding amino acid sequence of either SEQ ID NO:2or SEQ ID NO:5 is due to a conservative amino acid substitution. Thepresent invention further contemplates isolated nucleic acid moleculesthat comprise a nucleotide sequence of nucleotides 132 to 389 of SEQ IDNO:1, nucleotides 147 to 389 of SEQ ID NO:1, and nucleotides 148 to 405of SEQ ID NO:4.

The present invention also includes vectors and expression vectorscomprising such nucleic acid molecules. Such expression vectors maycomprise a transcription promoter, and a transcription terminator,wherein the promoter is operably linked with the nucleic acid molecule,and wherein the nucleic acid molecule is operably linked with thetranscription terminator. The present invention further includesrecombinant host cells comprising these vectors and expression vectors.Illustrative host cells include bacterial, yeast, avian, fungal, insect,mammalian, and plant cells. Recombinant host cells comprising suchexpression vectors can be used to prepare Zven polypeptides by culturingsuch recombinant host cells that comprise the expression vector and thatproduce the Zven protein, and, optionally, isolating the Zven proteinfrom the cultured recombinant host cells. The present invention furtherincludes products made by such processes.

In addition, the present invention provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and at least one ofsuch an expression vector or recombinant virus comprising suchexpression vectors.

The present invention also contemplates methods for detecting thepresence of Zven1 RNA in a biological sample, comprising the steps of(a) contacting a Zven1 nucleic acid probe under hybridizing conditionswith either (i) test RNA molecules isolated from the biological sample,or (ii) nucleic acid molecules synthesized from the isolated RNAmolecules, wherein the probe has a nucleotide sequence comprising aportion of the nucleotide sequence of SEQ ID NO:1, or its complement,and (b) detecting the formation of hybrids of the nucleic acid probe andeither the test RNA molecules or the synthesized nucleic acid molecules,wherein the presence of the hybrids indicates the presence of Zven1 RNAin the biological sample. Analogous methods can be used to detect thepresence of Zven2 RNA in a biological sample, wherein the probe has anucleotide sequence comprising a portion of the nucleotide sequence ofSEQ ID NO:4, or its complement.

The present invention further provides methods for detecting thepresence of Zven polypeptide in a biological sample, comprising thesteps of: (a) contacting the biological sample with an antibody or anantibody fragment that specifically binds with a polypeptide eitherconsisting of the amino acid sequence of SEQ ID NO:2 or consisting ofthe amino acid sequence of SEQ ID NO:5, wherein the contacting isperformed under conditions that allow the binding of the antibody orantibody fragment to the biological sample, and (b) detecting any of thebound antibody or bound antibody fragment. Such an antibody or antibodyfragment may further comprise a detectable label selected from the groupconsisting of radioisotope, fluorescent label, chemiluminescent label,enzyme label, bioluminescent label, and colloidal gold.

Illustrative biological samples include human tissue, such as an autopsysample, a biopsy sample, body fluids and digestive components, and thelike.

The present invention also provides kits for performing these detectionmethods. For example, a kit for detection of Zven1 gene expression maycomprise a container that comprises a nucleic acid molecule, wherein thenucleic acid molecule is selected from the group consisting of (a) anucleic acid molecule comprising the nucleotide sequence of nucleotides66 to 161 of SEQ ID NO:1, (b) a nucleic acid molecule comprising thenucleotide sequence of nucleotides 288 to 389 of SEQ ID NO:1, (c) anucleic acid molecule comprising the complement of the nucleotidesequence of nucleic acid molecules (a) or (b), (d) a nucleic acidmolecule that is a fragment of (a) consisting of at least eightnucleotides, (e) a nucleic acid molecule that is a fragment of (b)consisting of at least eight nucleotides, (f) a nucleic acid moleculethat is a fragment of (c) consisting of at least eight nucleotides, and(g) a nucleic acid molecule that is a fragment of or consists of thenucleic acid sequence as shown in SEQ ID NO: 12, 13, 15, 16, 17, 18, 19,20, 23, or 24. A kit for detection of Zven2 gene expression may comprisea container that comprises a nucleic acid molecule, wherein the nucleicacid molecule is selected from the group consisting of (a) a nucleicacid molecule comprising the nucleotide sequence of nucleotides 334 to405 of SEQ ID NO:4, (b) a nucleic acid molecule comprising thecomplement of the nucleotide sequence of (a), (c) a nucleic acidmolecule that is a fragment of (a) consisting of at least eightnucleotides, and (d) a nucleic acid molecule that is a fragment of (b)consisting of at least eight nucleotides. Such kits may also comprise asecond container that comprises one or more reagents capable ofindicating the presence of the nucleic acid molecule.

On the other hand, a kit for detection of Zven protein may comprise acontainer that comprises an antibody, or an antibody fragment, thatspecifically binds with a polypeptide consisting of the amino acidsequence of SEQ ID NO:2 or consisting of the amino acid sequence of SEQID NO:5.

The present invention also contemplates anti-idiotype antibodies, oranti-idiotype antibody fragments, that specifically bind an antibody orantibody fragment that specifically binds a polypeptide consisting ofthe amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQID NO:5.

The present invention further provides variant Zven1 polypeptides, whichcomprise an amino acid sequence that shares an identity with the aminoacid sequence of SEQ ID NO:2 selected from the group consisting of atleast 70% identity, at least 80% identity, at least 90% identity, atleast 95% identity, or greater than 95% identity, and wherein anydifference between the amino acid sequence of the variant polypeptideand the amino acid sequence of SEQ ID NO:2 is due to one or moreconservative amino acid substitutions. Illustrative variant Zven2polypeptides, which comprise an amino acid sequence that shares anidentity with the amino acid sequence of SEQ ID NO:5 selected from thegroup consisting of at least 70% identity, at least 80% identity, atleast 90% identity, at least 95% identity, or greater than 95% identity,and wherein any difference between the amino acid sequence of thevariant polypeptide and the amino acid sequence of SEQ ID NO:5 is due toone or more conservative amino acid substitutions.

The present invention also provides fusion proteins comprising a Zven1polypeptide moiety or a Zven2 polypeptide moiety. Such fusion proteinscan further comprise an immunoglobulin moiety. A suitable immunoglobulinmoiety is an immunoglobulin heavy chain constant region, such as a humanFc fragment. The present invention also includes isolated nucleic acidmolecules that encode such fusion proteins.

The present invention further provides a method of treating defectiveileal contractility disease in a mammalian subject in need of suchtreatment, comprising: administering to the mammalian subject a Zven1polypeptide, wherein the Zven1 polypeptide comprises the amino acidsequence of amino acid residues 23 to 108 of SEQ ID NO:2. In oneembodiment, the disease is diabetes mellitus. In another method, thedisease is post-operative ileus. In another embodiment, the disease issepsis-related gastrointestinal stasis or ileus.

The present invention further provides a method of treating defectiveileal contractility disease in a mammalian subject in need of suchtreatment, comprising: administering to the mammalian subject a Zven1polypeptide, wherein the Zven1 polypeptide comprises the amino acidsequence of amino acid residues 28 to 108 of SEQ ID NO:2, the amino acidsequence of amino acid residues 20 to 105 of SEQ ID NO:5, or the aminoacid sequence of amino acid residues 28 to 129 of SEQ ID NO:29. In oneembodiment, the disease is diabetes mellitus. In another embodiment, thedisease is post-operative ileus. In another embodiment, the disease issepsis-related gastrointestinal stasis or ileus.

The invention further provides a method of modulating gastrointestinalcontractility in a mammal in need thereof comprising administering tothe mammal a polypeptide, wherein the polypeptide comprises the aminoacid sequence of amino acid residues 28 to 108 of SEQ ID NO:2. In anembodiment, the modulation is inhibition. In another embodiment, thepolypeptide is administered in one or more administrations. In a furtherembodiment, one or more of the administrations of the polypeptidestimulates gastrointestinal contractility, and wherein one or more ofthe administrations of the polypeptide inhibits gastrointestinalcontractility. In another embodiment, the one or more administrationsthat stimulate gastrointestinal contractility are administered beforethe one or more administrations that inhibit gastrointestinalcontractility. In another embodiment, the one or more administrationsthat inhibit gastrointestinal contractility are administered before theone or more administrations that stimulate gastrointestinalcontractility. In a further embodiment, therapeutic control of gastriccontractility is achieved. In another embodiment, the polypeptide isadministered continually for a period of time.

The invention also provides a method of stimulating gastrointestinalcontractility comprising administering to a mammal in need thereof apolypeptide comprising the amino acid sequence of amino acid residuesacids 20 to 105 as shown in SEQ ID NO:5, followed by administering apolypeptide comprising the amino acid sequence of amino acids 28 to 108of SEQ ID NO:2.

The invention also provides a method of modulating gastric emptying in amammal in need thereof comprising administering to the mammal apolypeptide, wherein the polypeptide comprises the amino acid sequenceof amino acid residues 28 to 108 of SEQ ID NO:2. In an embodiment, themodulation is inhibition. In another embodiment, the polypeptide isadministered in one or more administration. In another embodiment, oneor more of the administrations of the polypeptide stimulates gastricemptying, and wherein one or more of the administrations of thepolypeptide inhibits gastric emptying. In a further embodiment, the oneor more administrations that stimulate gastric emptying are administeredbefore the one or more administrations that inhibit gastric emptying. Inanother further embodiment, the one or more administrations that inhibitgastric emptying are administered before the one or more administrationsthat stimulate gastric emptying. In another embodiment, therapeuticcontrol of gastric emptying is achieved. In another embodiment thepolypeptide is administered continually for a period of time.

The invention also provides a method of modulating intestinal transit ina mammal in need thereof comprising administering to the mammal apolypeptide, wherein the polypeptide comprises the amino acid sequenceof amino acid residues 28 to 108 of SEQ ID NO:2. In an embodiment, themodulation is inhibition. In another embodiment, the polypeptide isadministered in one or more administration. In a further embodiment, oneor more of the administrations of the polypeptide stimulates intestinaltransit, and wherein one or more of the administrations of thepolypeptide are effective in inhibiting intestinal transit. In a furtherembodiment, the one or more administrations that stimulate intestinaltransit are administered before the one or more administrations thatinhibit intestinal transit. In another further embodiment, the one ormore administrations that inhibit intestinal transit are administeredbefore the one or more administrations that stimulate intestinaltransit. In another embodiment, therapeutic control of intestinaltransit is achieved. In another embodiment, the polypeptide isadministered continually for a period of time.

The invention also provides a method of treating gastroparesis in amammal in thereof comprising, administering to the mammal a polypeptide,wherein the polypeptide comprises the amino acid sequence of amino acidresidues 28 to 108 of SEQ ID NO:2, and wherein gastrointestinalcontractility, gastric emptying, or intestinal transit is improved. Inan embodiment, the gastroparesis is related to surgery. In anotherembodiment, the polypeptide is administered to the mammal before orafter the surgery. In another embodiment, the polypeptide isadministered to the mammal before or after the mammal is fed apost-surgery meal. In another embodiment, the treatment is characterizedby an increase in contractility in the ileus. In another embodiment, thegastroparesis is post-operative ileus, or paralytic ileus. In anotherembodiment, the gastroparesis is not related to surgery. In anotherembodiment, the gastroparesis is related to diabetes, intestinalpseudo-obstruction, chronic constipation, dyspepsia, gastroesophagealreflux, emesis, paralytic gastroparesis, sepsis, or consumption ofmedications.

The invention also provides a method of stimulating chemokine release ina mammal in need thereof, comprising administering to the mammal apolypeptide, wherein the polypeptide comprises the amino acid sequenceof amino acid residues 28 to 108 of SEQ ID NO:2 or the amino acidsequence of amino acid residues 20 to 105 of SEQ ID NO:5.

The invention also provides a method of stimulating chemokine release ina mammal in need thereof, comprising administering to the mammal apolypeptide, wherein the polypeptide comprises the amino acid sequenceof amino acid residues 28 to 108 of SEQ ID NO:2 or the amino acidsequence of amino acid residues 20 to 105 of SEQ ID NO:5.

The invention also provides a method of stimulating neutrophilinfiltration in a mammal in need thereof, comprising administering tothe mammal a polypeptide, wherein the polypeptide comprises the aminoacid sequence of amino acid residues 28 to 108 of SEQ ID NO:2 or theamino acid sequence of amino acid residues 20 to 105 of SEQ ID NO:5.

The invention also provides a method of inducing or increasing appetiteor weight gain in a mammal in need thereof comprising administering tothe mammal a polypeptide, wherein the polypeptide comprises the aminoacid sequence of amino acid residues 28 to 108 of SEQ ID NO:2 or theamino acid sequence of amino acid residues 20 to 105 of SEQ ID NO:5.

The invention also provides a method of increasing sensitization to athermal, mechanical or painful stimulus in a mammal in need thereof,comprising administering to the mammal a polypeptide, wherein thepolypeptide comprises the amino acid sequence of amino acid residues 28to 108 of SEQ ID NO:2 or the amino acid sequence of amino acid residues20 to 105 of SEQ ID NO:5.

The invention also provides a method of decreasing sensitization to athermal, mechanical or painful stimulus in a mammal in need thereof,comprising administering to the mammal an antagonist to a polypeptide,wherein the polypeptide comprises the amino acid sequence of amino acidresidues 28 to 108 of SEQ ID NO:2 or the amino acid sequence of aminoacid residues 20 to 105 of SEQ ID NO:5. Within an embodiment, theantagonist is an antibody that specifically binds to the polypeptide.

The invention also provides a method for inducing vasculogenesis incardiac stem cells, comprising administering a polypeptide, wherein thepolypeptide comprises the amino acid sequence of amino acid residues 28to 108 of SEQ ID NO:2. Within an embodiment, the vasculogenesis isinduced ex vivo or in vitro.

The invention also provides a for inducing angiogenesis in cardiac stemcells, comprising administering a polypeptide, wherein the polypeptidecomprises the amino acid sequence of amino acid residues 28 to 108 ofSEQ ID NO:2. Within an embodiment, the neogenesis is induced ex vivo orin vitro.

The invention also provides a method of modulating gastrointestinalcontractility, gastric emptying or intestinal transit in a mammal inneed thereof, comprising administering to the mammal a polypeptide,wherein the polypeptide comprises the amino acid sequence selected from:amino acid residues 28 to 108 of SEQ ID NO:2; amino acid residues 20 to105 of SEQ ID NO:5; and amino acid residues 28 to 129 of SEQ ID NO:29.Within an embodiment, the polypeptide is administered orally,intraperitoneally, intravenously, intramuscularly, or subcutaneously.

The invention also provides an isolated nucleic acid comprising thenucleic acid sequence as shown in SEQ ID NO:14.

The invention also provides a method of producing a polypeptide,comprising the step of culturing recombinant host cells that comprise anexpression vector, wherein the expression vector comprises the isolatednucleic acid of as shown in SEQ ID NO:14, a transcription promoter, anda transcription terminator, wherein the promoter is operably linked withthe nucleic acid, and wherein the nucleic acid is operably linked withthe transcription terminator, and wherein the protein encoded by thenucleic acid is produced by the recombinant cell. The invention alsoprovides the polypeptide produced by the method.

These and other aspects of the invention will become evident uponreference to the following detailed description. In addition, variousreferences are identified below and are incorporated by reference intheir entirety.

2. Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “contig” denotes a nucleic acid molecule that has a contiguousstretch of identical or complementary sequence to another nucleic acidmolecule. Contiguous sequences are said to “overlap” a given stretch ofa nucleic acid molecule either in their entirety or along a partialstretch of the nucleic acid molecule.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol. 1:47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response elementbinding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre and Rousseau, Biochem. J. 303:1 (1994)). If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

An “integrated genetic element” is a segment of DNA that has beenincorporated into a chromosome of a host cell after that element isintroduced into the cell through human manipulation. Within the presentinvention, integrated genetic elements are most commonly derived fromlinearized plasmids that are introduced into the cells byelectroporation or other techniques. Integrated genetic elements arepassed from the original host cell to its progeny.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that produces aZven1 or Zven2 peptide or polypeptide from an expression vector. Incontrast, such polypeptides can be produced by a cell that is a “naturalsource” of Zven1 or Zven2, and that lacks an expression vector.

“Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least two genes. Forexample, a fusion protein can comprise at least part of a Zven1 or Zven2polypeptide fused with a polypeptide that binds an affinity matrix. Sucha fusion protein provides a means to isolate large quantities of Zven1or Zven2 using affinity chromatography.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

The term “secretory signal sequence” denotes a DNA sequence that encodesa peptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure, orgreater than 99% pure. One way to show that a particular proteinpreparation contains an isolated polypeptide is by the appearance of asingle band following sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis of the protein preparation and Coomassie Brilliant Bluestaining of the gel. However, the term “isolated” does not exclude thepresence of the same polypeptide in alternative physical forms, such asdimers or alternatively glycosylated or derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a polypeptide encoded by asplice variant of an mRNA transcribed from a gene.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, co-stimulatory molecules, hematopoieticfactors, and synthetic analogs of these molecules.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

An “anti-idiotype antibody” is an antibody that binds with the variableregion domain of an immunoglobulin. In the present context, ananti-idiotype antibody binds with the variable region of an anti-Zven1or anti-Zven2 antibody, and thus, an anti-idiotype antibody mimics anepitope of Zven1 or Zven2.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. For example, an anti-Zven1 monoclonal antibody fragment bindswith an epitope of Zven1.

The term “antibody fragment” also includes a synthetic or a geneticallyengineered polypeptide that binds to a specific antigen, such aspolypeptides consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

A “chimeric antibody” is a recombinant protein that contains thevariable domains and complementary determining regions derived from arodent antibody, while the remainder of the antibody molecule is derivedfrom a human antibody.

“Humanized antibodies” are recombinant proteins in which murinecomplementarity determining regions of a monoclonal antibody have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain.

A “detectable label” is a molecule or atom which can be conjugated to anantibody moiety to produce a molecule useful for diagnosis. Examples ofdetectable labels include chelators, photoactive agents, radioisotopes,fluorescent agents, paramagnetic ions, or other marker moieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNAs encoding affinity tags are available fromcommercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

A “naked antibody” is an entire antibody, as opposed to an antibodyfragment, which is not conjugated with a therapeutic agent. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric and humanizedantibodies.

As used herein, the term “antibody component” includes both an entireantibody and an antibody fragment.

A “target polypeptide” or a “target peptide” is an amino acid sequencethat comprises at least one epitope, and that is expressed on a targetcell, such as a tumor cell, or a cell that carries an infectious agentantigen. T cells recognize peptide epitopes presented by a majorhistocompatibility complex molecule to a target polypeptide or targetpeptide and typically lyse the target cell or recruit other immune cellsto the site of the target cell, thereby killing the target cell.

An “antigenic peptide” is a peptide, which will bind a majorhistocompatibility complex molecule to form an MHC-peptide complex whichis recognized by a T cell, thereby inducing a cytotoxic lymphocyteresponse upon presentation to the T cell. Thus, antigenic peptides arecapable of binding to an appropriate major histocompatibility complexmolecule and inducing a cytotoxic T cells response, such as cell lysisor specific cytokine release against the target cell which binds orexpresses the antigen. The antigenic peptide can be bound in the contextof a class I or class II major histocompatibility complex molecule, onan antigen presenting cell or on a target cell.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

An “anti-sense oligonucleotide specific for Zven1” or a “Zven1anti-sense oligonucleotide” is an oligonucleotide having a sequence (a)capable of forming a stable triplex with a portion of the Zven1 gene, or(b) capable of forming a stable duplex with a portion of an mRNAtranscript of the Zven1 gene. Similarly, an “anti-sense oligonucleotidespecific for Zven2” or a “Zven2 anti-sense oligonucleotide” is anoligonucleotide having a sequence (a) capable of forming a stabletriplex with a portion of the Zven2 gene, or (b) capable of forming astable duplex with a portion of an mRNA transcript of the Zven2 gene.

A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

An “external guide sequence” is a nucleic acid molecule that directs theendogenous ribozyme, RNase P, to a particular species of intracellularmRNA, resulting in the cleavage of the mRNA by RNase P. A nucleic acidmolecule that encodes an external guide sequence is termed an “externalguide sequence gene.”

The term “variant Zven1 gene” refers to nucleic acid molecules thatencode a polypeptide having an amino acid sequence that is amodification of SEQ ID NO:2. Such variants include naturally-occurringpolymorphisms of Zven1 genes, as well as synthetic genes that containconservative amino acid substitutions of the amino acid sequence of SEQID NO:2. Additional variant forms of Zven1 genes are nucleic acidmolecules that contain insertions or deletions of the nucleotidesequences described herein. A variant Zven1 gene can be identified bydetermining whether the gene hybridizes with a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:1, or its complement, understringent conditions. Similarly, a variant Zven2 gene and a variantZven2 polypeptide can be identified with reference to SEQ ID NO:4 andSEQ ID NO:5, respectively.

Alternatively, variant Zven genes can be identified by sequencecomparison. Two amino acid sequences have “100% amino acid sequenceidentity” if the amino acid residues of the two amino acid sequences arethe same when aligned for maximal correspondence. Similarly, twonucleotide sequences have “100% nucleotide sequence identity” if thenucleotide residues of the two nucleotide sequences are the same whenaligned for maximal correspondence. Sequence comparisons can beperformed using standard software programs such as those included in theLASERGENE bioinformatics computing suite, which is produced by DNASTAR(Madison, Wis.). Other methods for comparing two nucleotide or aminoacid sequences by determining optimal alignment are well-known to thoseof skill in the art (see, for example, Peruski and Peruski, The Internetand the New Biology: Tools for Genomic and Molecular Research (ASMPress, Inc. 1997), Wu et al. (eds.), “Information Superhighway andComputer Databases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology, pages 123-151 (CRC Press, Inc. 1997), and Bishop (ed.),Guide to Human Genome Computing, 2nd Edition (Academic Press, Inc.1998)). Particular methods for determining sequence identity aredescribed below.

Regardless of the particular method used to identify a variant Zven1gene or variant Zven1 polypeptide, a variant gene or polypeptide encodedby a variant gene may be characterized by its ability to bindspecifically to an anti-Zven1 antibody. Similarly, a variant Zven2 geneproduct or variant Zven2 polypeptide may be characterized by its abilityto bind specifically to an anti-Zven2 antibody.

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

The present invention includes functional fragments of Zven1 and Zven2genes. Within the context of this invention, a “functional fragment” ofa Zven1 (or Zven2) gene refers to a nucleic acid molecule that encodes aportion of a Zven1 (or Zven2) polypeptide, which specifically binds withan anti-Zven1 (anti-Zven2) antibody.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to 110%.

3. Production of Human Zven1 and Zven2 Genes

Nucleic acid molecules encoding a human Zven1 gene can be obtained byscreening a human cDNA or genomic library using polynucleotide probesbased upon SEQ ID NO:1. Similarly, nucleic acid molecules encoding ahuman Zven2 gene can be obtained by screening a human cDNA or genomiclibrary using polynucleotide probes based upon SEQ ID NO:4. Thesetechniques are standard and well-established.

As an illustration, a nucleic acid molecule that encodes a human Zven1gene can be isolated from a human cDNA library. In this case, the firststep would be to prepare the cDNA library by isolating RNA from tissues,such as testis or peripheral blood lymphocytes, using methods well-knownto those of skill in the art. In general, RNA isolation techniques mustprovide a method for breaking cells, a means of inhibitingRNase-directed degradation of RNA, and a method of separating RNA fromDNA, protein, and polysaccharide contaminants. For example, total RNAcan be isolated by freezing tissue in liquid nitrogen, grinding thefrozen tissue with a mortar and pestle to lyse the cells, extracting theground tissue with a solution of phenol/chloroform to remove proteins,and separating RNA from the remaining impurities by selectiveprecipitation with lithium chloride (see, for example, Ausubel et al.(eds.), Short Protocols in Molecular Biology, 3^(rd) Edition, pages 4-1to 4-6 (John Wiley & Sons 1995) [“Ausubel (1995)”]; Wu et al., Methodsin Gene Biotechnology, pages 33-41 (CRC Press, Inc. 1997) [“Wu(1997)”]). Alternatively, total RNA can be isolated from tissue byextracting ground tissue with guanidinium isothiocyanate, extractingwith organic solvents, and separating RNA from contaminants usingdifferential centrifugation (see, for example, Chirgwin et al.,Biochemistry 18:52 (1979); Ausubel (1995) at pages 4-1 to 4-6; Wu (1997)at pages 33-41).

In order to construct a cDNA library, poly(A)⁺RNA must be isolated froma total RNA preparation. Poly(A)⁺ RNA can be isolated from total RNAusing the standard technique of oligo(dT)-cellulose chromatography (see,for example, Aviv and Leder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972);Ausubel (1995) at pages 4-11 to 4-12).

Double-stranded cDNA molecules are synthesized from poly(A)+ RNA usingtechniques well-known to those in the art. (see, for example, Wu (1997)at pages 41-46). Moreover, commercially available kits can be used tosynthesize double-stranded cDNA molecules. For example, such kits areavailable from Life Technologies, Inc. (Gaithersburg, Md.), CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Promega Corporation (Madison,Wis.) and STRATAGENE (La Jolla, Calif.).

Various cloning vectors are appropriate for the construction of a cDNAlibrary. For example, a cDNA library can be prepared in a vector derivedfrom bacteriophage, such as a λgt10 vector. See, for example, Huynh etal., “Constructing and Screening cDNA Libraries in λgt10 and λ gt11,” inDNA Cloning: A Practical Approach Vol. 1, Glover (ed.), page 49 (IRLPress, 1985); Wu (1997) at pages 47-52.

Alternatively, double-stranded cDNA molecules can be inserted into aplasmid vector, such as a PBLUESCRIPT vector (STRATAGENE; La Jolla,Calif.), a LAMDAGEM-4 (Promega Corp.) or other commercially availablevectors. Suitable cloning vectors also can be obtained from the AmericanType Culture Collection (Manassas, Va.).

To amplify the cloned cDNA molecules, the cDNA library is inserted intoa prokaryotic host, using standard techniques. For example, a cDNAlibrary can be introduced into competent E. coli DH5 cells, which can beobtained, for example, from Life Technologies, Inc. (Gaithersburg, Md.).

A human genomic library can be prepared by means well-known in the art(see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) atpages 307-327). Genomic DNA can be isolated by lysing tissue with thedetergent Sarkosyl, digesting the lysate with proteinase K, clearinginsoluble debris from the lysate by centrifugation, precipitatingnucleic acid from the lysate using isopropanol, and purifyingresuspended DNA on a cesium chloride density gradient.

DNA fragments that are suitable for the production of a genomic librarycan be obtained by the random shearing of genomic DNA or by the partialdigestion of genomic DNA with restriction endonucleases. Genomic DNAfragments can be inserted into a vector, such as a bacteriophage orcosmid vector, in accordance with conventional techniques, such as theuse of restriction enzyme digestion to provide appropriate termini, theuse of alkaline phosphatase treatment to avoid undesirable joining ofDNA molecules, and ligation with appropriate ligases. Techniques forsuch manipulation are well-known in the art (see, for example, Ausubel(1995) at pages 5-1 to 5-6; Wu (1997) at pages 307-327).

Nucleic acid molecules that encode a human Zven1 or Zven2 gene can alsobe obtained using the polymerase chain reaction (PCR) witholigonucleotide primers having nucleotide sequences that are based uponthe nucleotide sequences described herein. General methods for screeninglibraries with PCR are provided by, for example, Yu et al., “Use of thePolymerase Chain Reaction to Screen Phage Libraries,” in Methods inMolecular Biology, Vol. 15: PCR Protocols: Current Methods andApplications, White (ed.), pages 211-215 (Humana Press, Inc. 1993).Moreover, techniques for using PCR to isolate related genes aredescribed by, for example, Preston, “Use of Degenerate OligonucleotidePrimers and the Polymerase Chain Reaction to Clone Gene Family Members,”in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methodsand Applications, White (ed.), pages 317-337 (Humana Press, Inc. 1993).

Alternatively, human genomic libraries can be obtained from commercialsources such as Research Genetics (Huntsville, Ala.) and the AmericanType Culture Collection (Manassas, Va.).

A library containing cDNA or genomic clones can be screened with one ormore polynucleotide probes based upon SEQ ID NO:1, using standardmethods (see, for example, Ausubel (1995) at pages 6-1 to 6-11).

Anti-Zven antibodies, produced as described below, can also be used toisolate DNA sequences that encode human Zven genes from cDNA libraries.For example, the antibodies can be used to screen λgt1 expressionlibraries, or the antibodies can be used for immunoscreening followinghybrid selection and translation (see, for example, Ausubel (1995) atpages 6-12 to 6-16; Margolis et al., “Screening X expression librarieswith antibody and protein probes,” in DNA Cloning 2: Expression Systems,2nd Edition, Glover et al. (eds.), pages 1-14 (Oxford University Press1995)).

As an alternative, a Zven gene can be obtained by synthesizing nucleicacid molecules using mutually priming long oligonucleotides and thenucleotide sequences described herein (see, for example, Ausubel (1995)at pages 8-8 to 8-9). Established techniques using the polymerase chainreaction provide the ability to synthesize DNA molecules at least twokilobases in length (Adang et al., Plant Molec. Biol. 21:1131 (1993),Bambot et al., PCR Methods and Applications 2:266 (1993), Dillon et al.,“Use of the Polymerase Chain Reaction for the Rapid Construction ofSynthetic Genes,” in Methods in Molecular Biology, Vol. 15: PCRProtocols: Current Methods and Applications, White (ed.), pages 263-268,(Humana Press, Inc. 1993), and Holowachuk et al., PCR Methods Appl.4:299 (1995)).

The nucleic acid molecules of the present invention can also besynthesized with “gene machines” using protocols such as thephosphoramidite method. If chemically-synthesized double stranded DNA isrequired for an application such as the synthesis of a gene or a genefragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 base pairs) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. For the production oflonger genes (>300 base pairs), however, special strategies may berequired, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length. For reviews onpolynucleotide synthesis, see, for example, Glick and Pasternak,Molecular Biotechnology, Principles and Applications of Recombinant DNA(ASM Press 1994), Itakura et al., Ann. Rev. Biochem. 53:323 (1984), andClimie et al., Proc. Nat'l Acad. Sci. USA 87:633 (1990).

The sequence of a Zven cDNA or Zven genomic fragment can be determinedusing standard methods. Zven polynucleotide sequences disclosed hereincan also be used as probes or primers to clone 5′ non-coding regions ofa Zven gene. Promoter elements from a Zven gene can be used to directthe expression of heterologous genes in tissues of, for example,transgenic animals or patients treated with gene therapy. Theidentification of genomic fragments containing a Zven promoter orregulatory element can be achieved using well-established techniques,such as deletion analysis (see, generally, Ausubel (1995)).

Cloning of 5′ flanking sequences also facilitates production of Zvenproteins by “gene activation,” as disclosed in U.S. Pat. No. 5,641,670.Briefly, expression of an endogenous Zven gene in a cell is altered byintroducing into the Zven locus a DNA construct comprising at least atargeting sequence, a regulatory sequence, an exon, and an unpairedsplice donor site. The targeting sequence is a Zven 5′ non-codingsequence that permits homologous recombination of the construct with theendogenous Zven locus, whereby the sequences within the construct becomeoperably linked with the endogenous Zven coding sequence. In this way,an endogenous Zven promoter can be replaced or supplemented with otherregulatory sequences to provide enhanced, tissue-specific, or otherwiseregulated expression.

4. Production of Zven Gene Variants

The present invention provides a variety of nucleic acid molecules,including DNA and RNA molecules, which encode the Zven polypeptidesdisclosed herein. Those skilled in the art will readily recognize that,in view of the degeneracy of the genetic code, considerable sequencevariation is possible among these polynucleotide molecules. SEQ ID NOs:3and 6 are a degenerate nucleotide sequences that encompasses all nucleicacid molecules that encode the Zven polypeptides of SEQ ID NOs:2 and 5,respectively. Those skilled in the art will recognize that thedegenerate sequence of SEQ ID NO:3 also provides all RNA sequencesencoding SEQ ID NO:2, by substituting U for T, while the degeneratesequence of SEQ ID NO:6 also provides all RNA sequences encoding SEQ IDNO:5, by substituting U for T. Thus, the present invention contemplatesZven1 polypeptide-encoding nucleic acid molecules comprising nucleotide66 to nucleotide 389 of SEQ ID NO:1, and their RNA equivalents, as wellas Zven2 polypeptide-encoding nucleic acid molecules comprisingnucleotide 91 to nucleotide 405 of SEQ ID NO:4, and their RNAequivalents.

Table 1 sets forth the one-letter codes used within SEQ ID NOs:3 and 6to denote degenerate nucleotide positions. “Resolutions” are thenucleotides denoted by a code letter. “Complement” indicates the codefor the complementary nucleotide(s). For example, the code Y denoteseither C or T, and its complement R denotes A or G, A beingcomplementary to T, and G being complementary to C.

TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NOs:3 and 6, encompassing allpossible codons for a given amino acid, are set forth in Table 2.

TABLE 2 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding an amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NOs:2 and 5. Variant sequences can be readily testedfor functionality as described herein.

Different species can exhibit “preferential codon usage.” In general,see, Grantham et al., Nuc. Acids Res. 8:1893 (1980), Haas et al. Curr.Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355 (1981), Grosjean andFiers, Gene 18:199 (1982), Holm, Nuc. Acids Res. 14:3075 (1986),Ikemura, J. Mol. Biol. 158:573 (1982), Sharp and Matassi, Curr. Opin.Genet. Dev. 4:851 (1994), Kane, Curr. Opin. Biotechnol. 6:494 (1995),and Makrides, Microbiol. Rev. 60:512 (1996). As used herein, the term“preferential codon usage” or “preferential codons” is a term of artreferring to protein translation codons that are most frequently used incells of a certain species, thus favoring one or a few representativesof the possible codons encoding each amino acid (See Table 2). Forexample, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG,or ACT, but in mammalian cells ACC is the most commonly used codon; inother species, for example, insect cells, yeast, viruses or bacteria,different Thr codons may be preferential. Preferential codons for aparticular species can be introduced into the polynucleotides of thepresent invention by a variety of methods known in the art. Introductionof preferential codon sequences into recombinant DNA can, for example,enhance production of the protein by making protein translation moreefficient within a particular cell type or species. Therefore, thedegenerate codon sequences disclosed in SEQ ID NOs:3 and 6 serve astemplates for optimizing expression of polynucleotides in various celltypes and species commonly used in the art and disclosed herein.Sequences containing preferential codons can be tested and optimized forexpression in various species, and tested for functionality as disclosedherein.

The present invention further provides variant polypeptides and nucleicacid molecules that represent counterparts from other species(orthologs). These species include, but are not limited to mammalian,avian, amphibian, reptile, fish, insect and other vertebrate andinvertebrate species. Of particular interest are Zven polypeptides fromother mammalian species, including porcine, ovine, bovine, canine,feline, equine, and other primate polypeptides. Orthologs of human Zvencan be cloned using information and compositions provided by the presentinvention in combination with conventional cloning techniques. Forexample, a cDNA can be cloned using mRNA obtained from a tissue or celltype that expresses Zven. Suitable sources of mRNA can be identified byprobing northern blots with probes designed from the sequences disclosedherein. A library is then prepared from mRNA of a positive tissue orcell line.

A Zven-encoding cDNA molecule can then be isolated by a variety ofmethods, such as by probing with a complete or partial human cDNA orwith one or more sets of degenerate probes based on the disclosedsequences. A cDNA can also be cloned using the polymerase chain reactionwith primers designed from the representative human Zven sequencesdisclosed herein. Within an additional method, the cDNA library can beused to transform or transfect host cells, and expression of the cDNA ofinterest can be detected with an antibody to Zven polypeptide. Similartechniques can also be applied to the isolation of genomic clones.

Those skilled in the art will recognize that the sequences disclosed inSEQ ID NOs:1 and 4 represent single alleles of human Zven1 and Zven2,respectively, and that allelic variation and alternative splicing areexpected to occur. Allelic variants of this sequence can be cloned byprobing cDNA or genomic libraries from different individuals accordingto standard procedures. Allelic variants of the nucleotide sequencesshown in SEQ ID NOs:1 and 4, including those containing silent mutationsand those in which mutations result in amino acid sequence changes, arewithin the scope of the present invention, as are proteins which areallelic variants of SEQ ID NOs:2 and 5. cDNA molecules generated fromalternatively spliced mRNAs, which retain the properties of the Zvenpolypeptide are included within the scope of the present invention, asare polypeptides encoded by such cDNAs and mRNAs. Allelic variants andsplice variants of these sequences can be cloned by probing cDNA orgenomic libraries from different individuals or tissues according tostandard procedures known in the art.

Within certain embodiments of the invention, the isolated nucleic acidmolecules can hybridize under stringent conditions to nucleic acidmolecules comprising nucleotide sequences disclosed herein. For example,such nucleic acid molecules can hybridize under stringent conditions tonucleic acid molecules consisting of the nucleotide sequence of SEQ IDNO:1, to nucleic acid molecules consisting of the nucleotide sequence ofnucleotides 66 to 161 of SEQ ID NO:1, to nucleic acid moleculesconsisting of the nucleotide sequence of nucleotides 288 to 389 of SEQID NO:1, to nucleic acid molecules consisting of the nucleotide sequenceof SEQ ID NO:4, to nucleic acid molecules consisting of the nucleotidesequence of nucleotides 334 to 405 of SEQ ID NO:4, or to nucleic acidmolecules consisting of nucleotide sequences that are the complements ofsuch sequences. In general, stringent conditions are selected to beabout 5° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA,can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases. Stringent hybridization conditionsencompass temperatures of about 5-25° C. below the T_(m) of the hybridand a hybridization buffer having up to 1 M Na⁺. Higher degrees ofstringency at lower temperatures can be achieved with the addition offormamide which reduces the T_(m) of the hybrid about 1° C. for each 1%formamide in the buffer solution. Generally, such stringent conditionsinclude temperatures of 20-70° C. and a hybridization buffer containingup to 6×SSC and O-50% formamide. A higher degree of stringency can beachieved at temperatures of from 40-70° C. with a hybridization bufferhaving up to 4×SSC and from 0-50% formamide. Highly stringent conditionstypically encompass temperatures of 42-70° C. with a hybridizationbuffer having up to 1×SSC and 0-50% formamide. Different degrees ofstringency can be used during hybridization and washing to achievemaximum specific binding to the target sequence. Typically, the washesfollowing hybridization are performed at increasing degrees ofstringency to remove non-hybridized polynucleotide probes fromhybridized complexes.

The above conditions are meant to serve as a guide and it is well withinthe abilities of one skilled in the art to adapt these conditions foruse with a particular polypeptide hybrid. The T_(m) for a specifictarget sequence is the temperature (under defined conditions) at which50% of the target sequence will hybridize to a perfectly matched probesequence. Those conditions that influence the T_(m) include, the sizeand base pair content of the polynucleotide probe, the ionic strength ofthe hybridization solution, and the presence of destabilizing agents inthe hybridization solution. Numerous equations for calculating T_(m) areknown in the art, and are specific for DNA, RNA and DNA-RNA hybrids andpolynucleotide probe sequences of varying length (see, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(Cold Spring Harbor Press 1989); Ausubel et al., (eds.), CurrentProtocols in Molecular Biology (John Wiley and Sons, Inc. 1987); Bergerand Kimmel (eds.), Guide to Molecular Cloning Techniques, (AcademicPress, Inc. 1987); and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227(1990)). Sequence analysis software such as OLIGO 6.0 (LSR; Long Lake,Minn.) and Primer Premier 4.0 (Premier Biosoft International; Palo Alto,Calif.), as well as sites on the Internet, are available tools foranalyzing a given sequence and calculating T_(m) based on user definedcriteria. Such programs can also analyze a given sequence under definedconditions and identify suitable probe sequences. Typically,hybridization of longer polynucleotide sequences, >50 base pairs, isperformed at temperatures of about 20-25° C. below the calculated T_(m).For smaller probes, <50 base pairs, hybridization is typically carriedout at the T_(m) or 5-10° C. below. This allows for the maximum rate ofhybridization for DNA-DNA and DNA-RNA hybrids.

The length of the polynucleotide sequence influences the rate andstability of hybrid formation. Smaller probe sequences, <50 base pairs,reach equilibrium with complementary sequences rapidly, but may formless stable hybrids. Incubation times of anywhere from minutes to hourscan be used to achieve hybrid formation. Longer probe sequences come toequilibrium more slowly, but form more stable complexes even at lowertemperatures. Incubations are allowed to proceed overnight or longer.Generally, incubations are carried out for a period equal to three timesthe calculated Cot time. Cot time, the time it takes for thepolynucleotide sequences to reassociate, can be calculated for aparticular sequence by methods known in the art.

The base pair composition of polynucleotide sequence will effect thethermal stability of the hybrid complex, thereby influencing the choiceof hybridization temperature and the ionic strength of the hybridizationbuffer. A-T pairs are less stable than G-C pairs in aqueous solutionscontaining sodium chloride. Therefore, the higher the G-C content, themore stable the hybrid. Even distribution of G and C residues within thesequence also contribute positively to hybrid stability. In addition,the base pair composition can be manipulated to alter the T_(m) of agiven sequence. For example, 5-methyldeoxycytidine can be substitutedfor deoxycytidine and 5-bromodeoxuridine can be substituted forthymidine to increase the T_(m), whereas 7-deazz-2′-deoxyguanosine canbe substituted for guanosine to reduce dependence on T_(m).

The ionic concentration of the hybridization buffer also affects thestability of the hybrid. Hybridization buffers generally containblocking agents such as Denhardt's solution (Sigma Chemical Co., St.Louis, Mo.), denatured salmon sperm DNA, tRNA, milk powders (BLOTTO),heparin or SDS, and a Na⁺ source, such as SSC (1×SSC: 0.15 M sodiumchloride, 15 mM sodium citrate) or SSPE (1×SSPE: 1.8 M NaCl, 10 mMNaH₂PO₄, 1 mM EDTA, pH 7.7). By decreasing the ionic concentration ofthe buffer, the stability of the hybrid is increased. Typically,hybridization buffers contain from between 10 mM-1 M Na⁺. The additionof destabilizing or denaturing agents such as formamide,tetralkylammonium salts, guanidinium cations or thiocyanate cations tothe hybridization solution will alter the T_(m) of a hybrid. Typically,formamide is used at a concentration of up to 50% to allow incubationsto be carried out at more convenient and lower temperatures. Formamidealso acts to reduce non-specific background when using RNA probes.

As an illustration, a nucleic acid molecule encoding a variant Zven1polypeptide can be hybridized with a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1 (or its complement) at 42° C.overnight in a solution comprising 50% formamide, 5×SSC (1×SSC: 0.15 Msodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution (100× Denhardt's solution: 2% (w/v) Ficoll400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin),10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA.One of skill in the art can devise variations of these hybridizationconditions. For example, the hybridization mixture can be incubated at ahigher temperature, such as about 65° C., in a solution that does notcontain formamide. Moreover, premixed hybridization solutions areavailable (e.g., EXPRESSHYB Hybridization Solution from CLONTECHLaboratories, Inc.), and hybridization can be performed according to themanufacturer's instructions.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×-2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 55-65° C. For example, nucleic acid moleculesencoding particular variant Zven1 polypeptides can remain hybridizedwith a nucleic acid molecule consisting of the nucleotide sequence ofnucleotides 66 to 161 of SEQ ID NO:1, the nucleotide sequence ofnucleotides 288 to 389 of SEQ ID NO:1, or their complements, followingwashing under stringent washing conditions, in which the wash stringencyis equivalent to 0.5×-2×SSC with 0.1% SDS at 55-65° C., including0.5×SSC with 0.1% SDS at 55° C., or 2×SSC with 0.1% SDS at 65° C. In asimilar manner, nucleic acid molecules encoding particular Zven2variants can remain hybridized with a nucleic acid molecule consistingof the nucleotide sequence of nucleotides 334 to 405 of SEQ ID NO:4, orits complement, following washing under stringent washing conditions, inwhich the wash stringency is equivalent to 0.5×-2×SSC with 0.1% SDS at55-65° C., including 0.5×SSC with 0.1% SDS at 55° C., or 2×SSC with 0.1%SDS at 65° C. One of skill in the art can readily devise equivalentconditions, for example, by substituting SSPE for SSC in the washsolution.

Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. As an illustration, nucleic acid molecules encoding particularvariant Zven1 polypeptides can remain hybridized with a nucleic acidmolecule consisting of the nucleotide sequence of nucleotides 66 to 161of SEQ ID NO:1, the nucleotide sequence of nucleotides 288 to 389 of SEQID NO:1, or their complements, following washing under highly stringentwashing conditions, in which the wash stringency is equivalent to0.1×-0.2×SSC with 0.1% SDS at 50-65° C., including 0.1×SSC with 0.1% SDSat 50° C., or 0.2×SSC with 0.1% SDS at 65° C. Similarly, nucleic acidmolecules encoding particular Zven2 variants remain hybridized with anucleic acid molecule consisting of the nucleotide sequence ofnucleotides 334 to 405 of SEQ ID NO:4, or its complement, followingwashing under highly stringent washing conditions, in which the washstringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C.,including 0.1×SSC with 0.1% SDS at 50° C., or 0.2×SSC with 0.1% SDS at65° C.

The present invention also provides isolated Zven1 polypeptides thathave a substantially similar sequence identity to the polypeptides ofSEQ ID NO:2, or their orthologs. The term “substantially similarsequence identity” is used herein to denote polypeptides having 85%,90%, 95% or greater than 95% sequence identity to the sequences shown inSEQ ID NO:2, or their orthologs. Similarly, the present inventionprovides isolated Zven2 polypeptides having 85%, 90%, 95% or greaterthan 95% sequence identity to the sequences shown in SEQ ID NO:5, ortheir orthologs.

The present invention also contemplates Zven variant nucleic acidmolecules that can be identified using two criteria: a determination ofthe similarity between the encoded polypeptide with the amino acidsequence of SEQ ID NOs:2 or 5, and a hybridization assay, as describedabove. For example, certain Zven1 gene variants include nucleic acidmolecules (1) that remain hybridized with a nucleic acid moleculeconsisting of the nucleotide sequence of nucleotides 66 to 161 of SEQ IDNO:1, the nucleotide sequence of nucleotides 288 to 389 of SEQ ID NO:1,or their complements, following washing under stringent washingconditions, in which the wash stringency is equivalent to 0.5×-2×SSCwith 0.1% SDS at 55-65° C., and (2) that encode a polypeptide having85%, 90%, 95% or greater than 95% sequence identity to the amino acidsequence of SEQ ID NO:2. Alternatively, certain Zven1 variant genes canbe characterized as nucleic acid molecules (1) that remain hybridizedwith a nucleic acid molecule consisting of the nucleotide sequence ofnucleotides 66 to 161 of SEQ ID NO:1, the nucleotide sequence ofnucleotides 288 to 389 of SEQ ID NO:1, or their complements, followingwashing under highly stringent washing conditions, in which the washstringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., and(2) that encode a polypeptide having 85%, 90%, 95% or greater than 95%sequence identity to the amino acid sequence of SEQ ID NO:2.

Moreover, certain Zven2 gene variants include nucleic acid molecules (1)that remain hybridized with a nucleic acid molecule consisting of thenucleotide sequence of nucleotides 334 to 405 of SEQ ID NO:4, or itscomplement, following washing under stringent washing conditions, inwhich the wash stringency is equivalent to 0.5×-2×SSC with 0.1% SDS at55-65° C., and (2) that encode a polypeptide having 85%, 90%, 95% orgreater than 95% sequence identity to the amino acid sequence of SEQ IDNO:5. Alternatively, certain Zven2 variant genes can be characterized asnucleic acid molecules (1) that remain hybridized with a nucleic acidmolecule consisting of the nucleotide sequence of nucleotides 334 to 405of SEQ ID NO:4, or its complement, following washing under highlystringent washing conditions, in which the wash stringency is equivalentto 0.1×-0.2×SSC with 0.1% SDS at 50-65° C., and (2) that encode apolypeptide having 85%, 90%, 95% or greater than 95% sequence identityto the amino acid sequence of SEQ ID NO:5.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 3 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as: ([Total number ofidentical matches]/[length of the longer sequence plus the number ofgaps introduced into the longer sequence in order to align the twosequences])(100).

TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativeZven1 or Zven2 variant. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NO:2) and a testsequence that have either the highest density of identities (if the ktupvariable is 1) or pairs of identities (if ktup=2), without consideringconservative amino acid substitutions, insertions, or deletions. The tenregions with the highest density of identities are then rescored bycomparing the similarity of all paired amino acids using an amino acidsubstitution matrix, and the ends of the regions are “trimmed” toinclude only those residues that contribute to the highest score. Ifthere are several regions with scores greater than the “cutoff” value(calculated by a predetermined formula based upon the length of thesequence and the ktup value), then the trimmed initial regions areexamined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62. These parameters can be introducedinto a FASTA program by modifying the scoring matrix file (“SMATRIX”),as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, and most preferably, three. The other parameters canbe set as: gap opening penalty=10, and gap extension penalty=1.

The present invention includes nucleic acid molecules that encode apolypeptide having a conservative amino acid change, compared with theamino acid sequence of SEQ ID NOs:2 or 5. That is, variants can beobtained that contain one or more amino acid substitutions of SEQ IDNOs:2 or 5, in which an alkyl amino acid is substituted for an alkylamino acid in a Zven1 or Zven2 amino acid sequence, an aromatic aminoacid is substituted for an aromatic amino acid in a Zven1 or Zven2 aminoacid sequence, a sulfur-containing amino acid is substituted for asulfur-containing amino acid in a Zven1 or Zven2 amino acid sequence, ahydroxy-containing amino acid is substituted for a hydroxy-containingamino acid in a Zven1 or Zven2 amino acid sequence, an acidic amino acidis substituted for an acidic amino acid in a Zven1 or Zven2 amino acidsequence, a basic amino acid is substituted for a basic amino acid in aZven1 or Zven2 amino acid sequence, or a dibasic monocarboxylic aminoacid is substituted for a dibasic monocarboxylic amino acid in a Zven1or Zven2 amino acid sequence.

Among the common amino acids, for example, a “conservative amino acidsubstitution” is illustrated by a substitution among amino acids withineach of the following groups: (1) glycine, alanine, valine, leucine, andisoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine andthreonine, (4) aspartate and glutamate, (5) glutamine and asparagine,and (6) lysine, arginine and histidine.

The BLOSUM62 table is an amino acid substitution matrix derived fromabout 2,000 local multiple alignments of protein sequence segments,representing highly conserved regions of more than 500 groups of relatedproteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915(1992)). Accordingly, the BLOSUM62 substitution frequencies can be usedto define conservative amino acid substitutions that may be introducedinto the amino acid sequences of the present invention. Although it ispossible to design amino acid substitutions based solely upon chemicalproperties (as discussed above), the language “conservative amino acidsubstitution” preferably refers to a substitution represented by aBLOSUM62 value of greater than −1. For example, an amino acidsubstitution is conservative if the substitution is characterized by aBLOSUM62 value of 0, 1, 2, or 3. According to this system, preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 1 (e.g., 1, 2 or 3), while more preferred conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 2 (e.g., 2 or 3).

Particular variants of Zven1 or Zven2 are characterized by having atleast 70%, at least 80%, at least 85%, at least 90%, at least 95% orgreater than 95% sequence identity to a corresponding amino acidsequence disclosed herein (i.e., SEQ ID NO:2 or SEQ ID NO:5), whereinthe variation in amino acid sequence is due to one or more conservativeamino acid substitutions.

Conservative amino acid changes in a Zven1 gene and a Zven2 gene can beintroduced by substituting nucleotides for the nucleotides recited inSEQ ID NO:1 and SEQ ID NO:4, respectively. Such “conservative aminoacid” variants can be obtained, for example, by oligonucleotide-directedmutagenesis, linker-scanning mutagenesis, mutagenesis using thepolymerase chain reaction, and the like (see Ausubel (1995) at pages8-10 to 8-22; and McPherson (ed.), Directed Mutagenesis: A PracticalApproach (IRL Press 1991)).

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is typicallycarried out in a cell-free system comprising an E. coli S30 extract andcommercially available enzymes and other reagents. Proteins are purifiedby chromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722 (1991), Ellman et al., Methods Enzymol. 202:301 (1991), Chunget al., Science 259:806 (1993), and Chung et al., Proc. Nat'l Acad. Sci.USA 90:10145 (1993).

In a second method, translation is carried out in Xenopus oocytes bymicroinjection of mutated mRNA and chemically aminoacylated suppressortRNAs (Turcatti et al., J. Biol. Chem. 271:19991 (1996)). Within a thirdmethod, E. coli cells are cultured in the absence of a natural aminoacid that is to be replaced (e.g., phenylalanine) and in the presence ofthe desired non-naturally occurring amino acid(s) (e.g.,2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or4-fluorophenylalanine). The non-naturally occurring amino acid isincorporated into the protein in place of its natural counterpart. See,Koide et al., Biochem. 33:7470 (1994). Naturally occurring amino acidresidues can be converted to non-naturally occurring species by in vitrochemical modification. Chemical modification can be combined withsite-directed mutagenesis to further expand the range of substitutions(Wynn and Richards, Protein Sci. 2:395 (1993)).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for Zven amino acidresidues.

Amino acid sequence analysis indicates that Zven1 and Zven2 shareseveral motifs. For example, one motif is “AVITGAC[DE][KR]D” (SEQ IDNO:8), wherein acceptable amino acids for a given position are indicatedwithin square brackets. This motif occurs in Zven1 at amino acidresidues 28 to 37 of SEQ ID NO:2, and in Zven2 at amino acid residues 20to 29 of SEQ ID NO:5. Another motif is“CHP[GL][ST][HR]KVPFFX[KR]RXHHTCPCLP” (SEQ ID NO:9), wherein acceptableamino acids for a given position are indicated within square brackets,and “X” can be any amino acid residue. This motif occurs in Zven1 atamino acid residues 68 to 90 in SEQ ID NO:2, and in Zven2 at amino acidresidues 60 to 82 of SEQ ID NO:5. The present invention includespeptides and polypeptides comprising these motifs.

Sequence analysis also indicated that Zven1 and Zven2 include variousconservative amino acid substitutions with respect to each other.Accordingly, particular Zven1 variants can be designed by modifying itssequence to include one or more amino acid substitutions correspondingwith the Zven2 sequence, while particular Zven2 variants can be designedby modifying its sequence to include one or more amino acidsubstitutions corresponding with the Zven1 sequence. Such variants canbe constructed using Table 4, which presents exemplary conservativeamino acid substitutions found in Zven1 and Zven2. Although Zven1 andZven2 variants can be designed with any number of amino acidsubstitutions, certain variants will include at least about X amino acidsubstitutions, wherein X is selected from the group consisting of 2, 5,7, 10, 12, 14, 16, 18, and 20.

TABLE 4 Zven1 Zven2 Amino acid Position Amino acid Position (SEQ ID NO:2) Amino acid (SEQ ID NO: 5) Amino acid 4 Leu 4 Ala 7 Ala 7 Val 9 Leu 9Ile 14 Leu 14 Val 35 Asp 27 Glu 36 Lys 28 Arg 42 Gly 34 Ala 48 Val 40Ile 50 Ile 42 Leu 52 Val 44 Leu 53 Lys 45 Arg 55 Ile 47 Leu 63 Lys 55Arg 66 Asp 58 Glu 71 Leu 63 Gly 72 Thr 64 Ser 73 Arg 65 His 80 Arg 72Lys 93 Ala 85 Leu 102 Phe 94 Tyr

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Nat'l Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity, such as the ability to bind to an antibody, to identify aminoacid residues that are critical to the activity of the molecule. Seealso, Hilton et al., J. Biol. Chem. 271:4699 (1996).

The location of Zven1 or Zven2 receptor binding domains can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,Science 255:306 (1992), Smith et al., J. Mol. Biol. 224:899 (1992), andWlodaver et al., FEBS Lett. 309:59 (1992). Moreover, Zven1 or Zven2labeled with biotin or FITC can be used for expression cloning of Zven1or Zven2 receptors.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53 (1988)) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152 (1989)). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner etal., U.S. Pat. No. 5,223,409, Huse, international publication No. WO92/06204, and region-directed mutagenesis (Derbyshire et al., Gene46:145 (1986), and Ner et al., DNA 7:127, (1988)).

Variants of the disclosed Zven1 or Zven2 nucleotide and polypeptidesequences can also be generated through DNA shuffling as disclosed byStemmer, Nature 370:389 (1994), Stemmer, Proc. Nat'l Acad. Sci. USA91:10747 (1994), and international publication No. WO 97/20078. Briefly,variant DNA molecules are generated by in vitro homologous recombinationby random fragmentation of a parent DNA followed by reassembly usingPCR, resulting in randomly introduced point mutations. This techniquecan be modified by using a family of parent DNA molecules, such asallelic variants or DNA molecules from different species, to introduceadditional variability into the process. Selection or screening for thedesired activity, followed by additional iterations of mutagenesis andassay provides for rapid “evolution” of sequences by selecting fordesirable mutations while simultaneously selecting against detrimentalchanges.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides, or polypeptidesthat bind with anti-Zven1 or anti-Zven2 antibodies, can be recoveredfrom the host cells and rapidly sequenced using modern equipment. Thesemethods allow the rapid determination of the importance of individualamino acid residues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

The present invention also includes “functional fragments” of Zven1 orZven2 polypeptides and nucleic acid molecules encoding such functionalfragments. Routine deletion analyses of nucleic acid molecules can beperformed to obtain functional fragments of a nucleic acid molecule thatencodes a Zven1 or Zven2 polypeptide. As an illustration, DNA moleculeshaving the nucleotide sequence of SEQ ID NO:1 can be digested with Bal31nuclease to obtain a series of nested deletions. The fragments are theninserted into expression vectors in proper reading frame, and theexpressed polypeptides are isolated and tested for the ability to bindanti-Zven antibodies. One alternative to exonuclease digestion is to useoligonucleotide-directed mutagenesis to introduce deletions or stopcodons to specify production of a desired fragment. Alternatively,particular fragments of a Zven gene can be synthesized using thepolymerase chain reaction.

Methods for identifying functional domains are well-known to those ofskill in the art. For example, studies on the truncation at either orboth termini of interferons have been summarized by Horisberger and DiMarco, Pharmac. Ther. 66:507 (1995). Moreover, standard techniques forfunctional analysis of proteins are described by, for example, Treuteret al., Molec. Gen. Genet. 240:113 (1993), Content et al., “Expressionand preliminary deletion analysis of the 42 kDa 2-5 A synthetase inducedby human interferon,” in Biological Interferon Systems, Proceedings ofISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72(Nijhoff 1987), Herschman, “The EGF Receptor,” in Control of Animal CellProliferation, Vol. 1, Boynton et al., (eds.) pages 169-199 (AcademicPress 1985), Coumailleau et al., J. Biol. Chem. 270:29270 (1995);Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi et al.,Biochem. Pharmacol. 50:1295 (1995), and Meisel et al., Plant Molec.Biol. 30:1 (1996).

The present invention also contemplates functional fragments of a Zven1or Zven2 gene that have amino acid changes, compared with the amino acidsequence of SEQ ID NO:2 or SEQ ID NO:5. A variant Zven gene can beidentified on the basis of structure by determining the level ofidentity with the particular nucleotide and amino acid sequencesdisclosed herein. An alternative approach to identifying a variant geneon the basis of structure is to determine whether a nucleic acidmolecule encoding a potential variant Zven1 or Zven2 gene can hybridizeto a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1or SEQ ID NO:4, as discussed above.

The present invention also provides polypeptide fragments or peptidescomprising an epitope-bearing portion of a Zven1 or Zven2 polypeptidedescribed herein. Such fragments or peptides may comprise an“immunogenic epitope,” which is a part of a protein that elicits anantibody response when the entire protein is used as an immunogen.Immunogenic epitope-bearing peptides can be identified using standardmethods (see, for example, Geysen et al., Proc. Nat'l Acad. Sci. USA81:3998 (1983)).

In contrast, polypeptide fragments or peptides may comprise an“antigenic epitope,” which is a region of a protein molecule to which anantibody can specifically bind. Certain epitopes consist of a linear orcontiguous stretch of amino acids, and the antigenicity of such anepitope is not disrupted by denaturing agents. It is known in the artthat relatively short synthetic peptides that can mimic epitopes of aprotein can be used to stimulate the production of antibodies againstthe protein (see, for example, Sutcliffe et al., Science 219:660(1983)). Accordingly, antigenic epitope-bearing peptides andpolypeptides of the present invention are useful to raise antibodiesthat bind with the polypeptides described herein.

Antigenic epitope-bearing peptides and polypeptides can contain at leastfour to ten amino acids, at least ten to fifteen amino acids, or about15 to about 30 amino acids of SEQ ID NOs:2 or 5. Such epitope-bearingpeptides and polypeptides can be produced by fragmenting a Zven1 orZven2 polypeptide, or by chemical peptide synthesis, as describedherein. Moreover, epitopes can be selected by phage display of randompeptide libraries (see, for example, Lane and Stephen, Curr. Opin.Immunol. 5:268 (1993), and Cortese et al., Curr. Opin. Biotechnol. 7:616(1996)). Standard methods for identifying epitopes and producingantibodies from small peptides that comprise an epitope are described,for example, by Mole, “Epitope Mapping,” in Methods in MolecularBiology, Vol. 10, Manson (ed.), pages 105-116 (The Humana Press, Inc.1992), Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages60-84 (Cambridge University Press 1995), and Coligan et al. (eds.),Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages9.4.1-9.4.11 (John Wiley & Sons 1997).

Regardless of the particular nucleotide sequence of a variant Zven1 orZven2 gene, the gene encodes a polypeptide that may be characterized byits ability to bind specifically to an anti-Zven1 or anti-Zven2antibody.

In addition to the uses described above, polynucleotides andpolypeptides of the present invention are useful as educational tools inlaboratory practicum kits for courses related to genetics and molecularbiology, protein chemistry, and antibody production and analysis. Due toits unique polynucleotide and polypeptide sequences, molecules of Zven1or Zven2 can be used as standards or as “unknowns” for testing purposes.For example, Zven1 or Zven2 polynucleotides can be used as an aid, suchas, for example, to teach a student how to prepare expression constructsfor bacterial, viral, or mammalian expression, including fusionconstructs, wherein Zven1 or Zven2 is the gene to be expressed; fordetermining the restriction endonuclease cleavage sites of thepolynucleotides; determining mRNA and DNA localization of Zven1 or Zven2polynucleotides in tissues (i.e., by northern and Southern blotting aswell as polymerase chain reaction); and for identifying relatedpolynucleotides and polypeptides by nucleic acid hybridization. As anillustration, students will find that PvuII digestion of a nucleic acidmolecule consisting of the nucleotide sequence of nucleotides 66 to 389of SEQ ID NO:1 provides two fragments of about 123 base pairs, and 201base pairs, whereas HaeIII digestion yields fragments of about 46 basepairs, and 278 base pairs.

Zven1 or Zven2 polypeptides can be used as an aid to teach preparationof antibodies; identifying proteins by western blotting; proteinpurification; determining the weight of expressed Zven1 or Zven2polypeptides as a ratio to total protein expressed; identifying peptidecleavage sites; coupling amino and carboxyl terminal tags; amino acidsequence analysis, as well as, but not limited to monitoring biologicalactivities of both the native and tagged protein (i.e., proteaseinhibition) in vitro and in vivo. For example, students will find thatdigestion of unglycosylated Zven1 with cyanogen bromide yields fourfragments having approximate molecular weights of 148, 4337, 1909, 2402,and 2939, whereas digestion of unglycosylated Zven1 with BNPS orNCS/urea yields fragments having approximate molecular weights of 5231,and 6444.

Zven1 or Zven2 polypeptides can also be used to teach analytical skillssuch as mass spectrometry, circular dichroism, to determineconformation, especially of the four alpha helices, x-raycrystallography to determine the three-dimensional structure in atomicdetail, nuclear magnetic resonance spectroscopy to reveal the structureof proteins in solution. For example, a kit containing Zven1 or Zven2can be given to the student to analyze. Since the amino acid sequencewould be known by the instructor, the protein can be given to thestudent as a test to determine the skills or develop the skills of thestudent, the instructor would then know whether or not the student hascorrectly analyzed the polypeptide. Since every polypeptide is unique,the educational utility of Zven1 or Zven2 would be unique unto itself.

The antibodies which bind specifically to Zven1 or Zven2 can be used asa teaching aid to instruct students how to prepare affinitychromatography columns to purify Zven1 or Zven2, cloning and sequencingthe polynucleotide that encodes an antibody and thus as a practicum forteaching a student how to design humanized antibodies. The Zven1 orZven2 gene, polypeptide, or antibody would then be packaged by reagentcompanies and sold to educational institutions so that the students gainskill in art of molecular biology. Because each gene and protein isunique, each gene and protein creates unique challenges and learningexperiences for students in a lab practicum. Such educational kitscontaining the Zven1 or Zven2 gene, polypeptide, or antibody areconsidered within the scope of the present invention.

For any Zven polypeptide, including variants and fusion proteins, one ofordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the information setforth in Tables 1 and 2 above. Moreover, those of skill in the art canuse standard software to devise Zven1 or Zven2 variants based upon thenucleotide and amino acid sequences described herein. Accordingly, thepresent invention includes a computer-readable medium encoded with adata structure that provides at least one of the following sequences:SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQID NO:6. Suitable forms of computer-readable media include magneticmedia and optically-readable media. Examples of magnetic media include ahard or fixed drive, a random access memory (RAM) chip, a floppy disk,digital linear tape (DLT), a disk cache, and a ZIP disk. Opticallyreadable media are exemplified by compact discs (e.g., CD-read onlymemory (ROM), CD-rewritable (RW), and CD-recordable), and digitalversatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).

5. Production of Zven Fusion Proteins

Fusion proteins of Zven can be used to express a Zven polypeptide orpeptide in a recombinant host, and to isolate expressed Zvenpolypeptides and peptides. One type of fusion protein comprises apeptide that guides a Zven polypeptide from a recombinant host cell. Todirect a Zven polypeptide into the secretory pathway of a eukaryotichost cell, a secretory signal sequence (also known as a signal peptide,a leader sequence, prepro sequence or pre sequence) is provided in theZven expression vector. While the secretory signal sequence may bederived from Zven1 or Zven2, a suitable signal sequence may also bederived from another secreted protein or synthesized de novo. Thesecretory signal sequence is operably linked to a Zven1- orZven2-encoding sequence such that the two sequences are joined in thecorrect reading frame and positioned to direct the newly synthesizedpolypeptide into the secretory pathway of the host cell. Secretorysignal sequences are commonly positioned 5′ to the nucleotide sequenceencoding the polypeptide of interest, although certain secretory signalsequences may be positioned elsewhere in the nucleotide sequence ofinterest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland etal., U.S. Pat. No. 5,143,830).

Although the secretory signal sequence of Zven1, Zven2, or anotherprotein produced by mammalian cells (e.g., tissue-type plasminogenactivator signal sequence, as described, for example, in U.S. Pat. No.5,641,655) is useful for expression of Zven1 or Zven2 in recombinantmammalian hosts, a yeast signal sequence is preferred for expression inyeast cells. Examples of suitable yeast signal sequences are thosederived from yeast mating phermone α-factor (encoded by the MFα1 gene),invertase (encoded by the SUC2 gene), or acid phosphatase (encoded bythe PHO5 gene). See, for example, Romanos et al., “Expression of ClonedGenes in Yeast,” in DNA Cloning 2: A Practical Approach, 2^(nd) Edition,Glover and Hames (eds.), pages 123-167 (Oxford University Press 1995).

In bacterial cells, it is often desirable to express a heterologousprotein as a fusion protein to decrease toxicity, increase stability,and to enhance recovery of the expressed protein. For example, Zven1 orZven2 can be expressed as a fusion protein comprising a glutathioneS-transferase polypeptide. Glutathione S-transferease fusion proteinsare typically soluble, and easily purifiable from E. coli lysates onimmobilized glutathione columns. In similar approaches, a Zven1 or Zven2fusion protein comprising a maltose binding protein polypeptide can beisolated with an amylose resin column, while a fusion protein comprisingthe C-terminal end of a truncated Protein A gene can be purified usingIgG-Sepharose. Established techniques for expressing a heterologouspolypeptide as a fusion protein in a bacterial cell are described, forexample, by Williams et al., “Expression of Foreign Proteins in E. coliUsing Plasmid Vectors and Purification of Specific PolyclonalAntibodies,” in DNA Cloning 2: A Practical Approach, 2^(nd) Edition,Glover and Hames (Eds.), pages 15-58 (Oxford University Press 1995). Inaddition, commercially available expression systems are available. Forexample, the PINPOINT Xa protein purification system (PromegaCorporation; Madison, Wis.) provides a method for isolating a fusionprotein comprising a polypeptide that becomes biotinylated duringexpression with a resin that comprises avidin.

Peptide tags that are useful for isolating heterologous polypeptidesexpressed by either prokaryotic or eukaryotic cells includepolyHistidine tags (which have an affinity for nickel-chelating resin),c-myc tags, calmodulin binding protein (isolated with calmodulinaffinity chromatography), substance P, the RYIRS tag (which binds withanti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which bindswith anti-FLAG antibodies). See, for example, Luo et al., Arch. Biochem.Biophys. 329:215 (1996), Morganti et al., Biotechnol. Appl. Biochem.23:67 (1996), and Zheng et al., Gene 186:55 (1997). Nucleic acidmolecules encoding such peptide tags are available, for example, fromSigma-Aldrich Corporation (St. Louis, Mo.).

Another form of fusion protein comprises a Zven1 or Zven2 polypeptideand an immunoglobulin heavy chain constant region, typically an Fcfragment, which contains two constant region domains and a hinge regionbut lacks the variable region. As an illustration, Chang et al., U.S.Pat. No. 5,723,125, describe a fusion protein comprising a humaninterferon and a human immunoglobulin Fc fragment. The C-terminal of theinterferon is linked to the N-terminal of the Fc fragment by a peptidelinker moiety. An example of a peptide linker is a peptide comprisingprimarily a T cell inert sequence, which is immunologically inert. Anexemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGGS (SEQ ID NO:7). In this fusion protein, a preferred Fc moiety is ahuman γ4 chain, which is stable in solution and has little or nocomplement activating activity. Accordingly, the present inventioncontemplates a Zven fusion protein that comprises a Zven1 or Zven2polypeptide moiety and a human Fc fragment, wherein the C-terminus ofthe Zven polypeptide moiety is attached to the N-terminus of the Fcfragment via a peptide linker, such as a peptide consisting of the aminoacid sequence of SEQ ID NO:7.

In another variation, a Zven1 or Zven2 fusion protein comprises an IgGsequence, a Zven polypeptide moiety covalently joined to the aminoterminal end of the IgG sequence, and a signal peptide that iscovalently joined to the amino terminal of the Zven polypeptide moiety,wherein the IgG sequence consists of the following elements in thefollowing order: a hinge region, a CH₂ domain, and a CH₃ domain.Accordingly, the IgG sequence lacks a CH₁ domain. The Zven polypeptidemoiety displays a Zven1 or Zven2 activity, such as the ability to bindwith a Zven1 or Zven2 receptor. This general approach to producingfusion proteins that comprise both antibody and nonantibody portions hasbeen described by LaRochelle et al., EP 742830 (WO 95/21258).

Fusion proteins comprising a Zven1 or Zven2 polypeptide moiety and an Fcmoiety can be used, for example, as an in vitro assay tool. For example,the presence of a Zven1 or Zven2 receptor in a biological sample can bedetected using these Zven1 or Zven2-antibody fusion proteins, in whichthe Zven moiety is used to target the cognate receptor, and amacromolecule, such as Protein A or anti-Fc antibody, is used to detectthe bound fusion protein-ligand complex. In addition, antibody-Zvenfusion proteins, comprising antibody variable domains, are useful astherapeutic proteins, in which the antibody moiety binds with a targetantigen, such as a tumor associated antigen.

Fusion proteins can be prepared by methods known to those skilled in theart by preparing each component of the fusion protein and chemicallyconjugating them. Alternatively, a polynucleotide encoding bothcomponents of the fusion protein in the proper reading frame can begenerated using known techniques and expressed by the methods describedherein. General methods for enzymatic and chemical cleavage of fusionproteins are described, for example, by Ausubel (1995) at pages 16-19 to16-25.

6. Production of Zven Polypeptides

The polypeptides of the present invention, including full-lengthpolypeptides, functional fragments, and fusion proteins, can be producedin recombinant host cells following conventional techniques. To expressa Zven1 or Zven2 gene, a nucleic acid molecule encoding the polypeptidemust be operably linked to regulatory sequences that controltranscriptional expression in an expression vector and then, introducedinto a host cell. In addition to transcriptional regulatory sequences,such as promoters and enhancers, expression vectors can includetranslational regulatory sequences and a marker gene, which is suitablefor selection of cells that carry the expression vector.

Expression vectors that are suitable for production of a foreign proteinin eukaryotic cells typically contain (1) prokaryotic DNA elementscoding for a bacterial replication origin and an antibiotic resistancemarker to provide for the growth and selection of the expression vectorin a bacterial host; (2) eukaryotic DNA elements that control initiationof transcription, such as a promoter; and (3) DNA elements that controlthe processing of transcripts, such as a transcriptiontermination/polyadenylation sequence. As discussed above, expressionvectors can also include nucleotide sequences encoding a secretorysequence that directs the heterologous polypeptide into the secretorypathway of a host cell. For example, a Zven1 expression vector maycomprise a Zven1 gene and a secretory sequence derived from a Zven1 geneor another secreted gene.

Zven1 or Zven2 proteins of the present invention may be expressed inmammalian cells. Examples of suitable mammalian host cells includeAfrican green monkey kidney cells (Vero; ATCC CRL 1587), human embryonickidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells(BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells(MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61;CHO DG44 [Chasin et al., Som. Cell. Molec. Genet. 12:555 1986]), ratpituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rathepatoma cells (H-4-II-E; ATCC CRL 1548) SV40-transformed monkey kidneycells (COS-1; ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCCCRL 1658).

For a mammalian host, the transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, in which the regulatorysignals are associated with a particular gene which has a high level ofexpression. Suitable transcriptional and translational regulatorysequences also can be obtained from mammalian genes, such as actin,collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer et al., J. Molec. Appl. Genet. 1:273 (1982)), the TKpromoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 earlypromoter (Benoist et al., Nature 290:304 (1981)), the Rous sarcoma viruspromoter (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777 (1982)), thecytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and themouse mammary tumor virus promoter (see, generally, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163-181 (John Wiley & Sons, Inc. 1996)).

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNApolymerase promoter, can be used to control Zven1 or Zven2 geneexpression in mammalian cells if the prokaryotic promoter is regulatedby a eukaryotic promoter (Zhou et al., Mol. Cell. Biol. 10:4529 (1990),and Kaufman et al., Nucl. Acids Res. 19:4485 (1991)).

An expression vector can be introduced into host cells using a varietyof standard techniques including calcium phosphate transfection,liposome-mediated transfection, microprojectile-mediated delivery,electroporation, and the like. The transfected cells can be selected andpropagated to provide recombinant host cells that comprise theexpression vector stably integrated in the host cell genome. Techniquesfor introducing vectors into eukaryotic cells and techniques forselecting such stable transformants using a dominant selectable markerare described, for example, by Ausubel (1995) and by Murray (ed.), GeneTransfer and Expression Protocols (Humana Press 1991).

For example, one suitable selectable marker is a gene that providesresistance to the antibiotic neomycin. In this case, selection iscarried out in the presence of a neomycin-type drug, such as G-418 orthe like. Selection systems can also be used to increase the expressionlevel of the gene of interest, a process referred to as “amplification.”Amplification is carried out by culturing transfectants in the presenceof a low level of the selective agent and then increasing the amount ofselective agent to select for cells that produce high levels of theproducts of the introduced genes. A suitable amplifiable selectablemarker is dihydrofolate reductase, which confers resistance tomethotrexate. Other drug resistance genes (e.g., hygromycin resistance,multi-drug resistance, puromycin acetyltransferase) can also be used.Alternatively, markers that introduce an altered phenotype, such asgreen fluorescent protein, or cell surface proteins such as CD4, CD8,Class I MHC, placental alkaline phosphatase may be used to sorttransfected cells from untransfected cells by such means as FACS sortingor magnetic bead separation technology.

Zven1 or Zven2 polypeptides can also be produced by cultured mammaliancells using a viral delivery system. Exemplary viruses for this purposeinclude adenovirus, herpesvirus, vaccinia virus and adeno-associatedvirus (AAV). Adenovirus, a double-stranded DNA virus, is currently thebest studied gene transfer vector for delivery of heterologous nucleicacid (for a review, see Becker et al., Meth. Cell Biol. 43:161 (1994),and Douglas and Curiel, Science & Medicine 4:44 (1997)). Advantages ofthe adenovirus system include the accommodation of relatively large DNAinserts, the ability to grow to high-titer, the ability to infect abroad range of mammalian cell types, and flexibility that allows usewith a large number of available vectors containing different promoters.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. An option is to delete theessential E1 gene from the viral vector, which results in the inabilityto replicate unless the E1 gene is provided by the host cell. Adenovirusvector-infected human 293 cells (ATCC Nos. CRL-1573, 45504, 45505), forexample, can be grown as adherent cells or in suspension culture atrelatively high cell density to produce significant amounts of protein(see Garnier et al., Cytotechnol. 15:145 (1994)).

Zven1 or Zven2 genes may also be expressed in other higher eukaryoticcells, such as avian, fungal, insect, yeast, or plant cells. Thebaculovirus system provides an efficient means to introduce cloned Zven1or Zven2 genes into insect cells. Suitable expression vectors are basedupon the Autographa californica multiple nuclear polyhedrosis virus(AcMNPV), and contain well-known promoters such as Drosophila heatshockprotein (hsp) 70 promoter, Autographa californica nuclearpolyhedrosis virus immediate-early gene promoter (ie-1) and the delayedearly 39K promoter, baculovirus p10 promoter, and the Drosophilametallothionein promoter. A second method of making recombinantbaculovirus utilizes a transposon-based system described by Luckow(Luckow, et al., J. Virol. 67:4566 (1993)). This system, which utilizestransfer vectors, is sold in the BAC-to-BAC kit (Life Technologies,Rockville, Md.). This system utilizes a transfer vector, PFASTBAC (LifeTechnologies) containing a Tn7 transposon to move the DNA encoding theZven polypeptide into a baculovirus genome maintained in E. coli as alarge plasmid called a “bacmid.” See, Hill-Perkins and Possee, J. Gen.Virol. 71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994),and Chazenbalk, and Rapoport, J. Biol. Chem. 270:1543 (1995). Inaddition, transfer vectors can include an in-frame fusion with DNAencoding an epitope tag at the C- or N-terminus of the expressed Zvenpolypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer et al.,Proc. Nat'l Acad. Sci. 82:7952 (1985)). Using a technique known in theart, a transfer vector containing a Zven1 or Zven2 gene is transformedinto E. coli, and screened for bacmids, which contain an interruptedlacZ gene indicative of recombinant baculovirus. The bacmid DNAcontaining the recombinant baculovirus genome is then isolated usingcommon techniques.

The illustrative PFASTBAC vector can be modified to a considerabledegree. For example, the polyhedrin promoter can be removed andsubstituted with the baculovirus basic protein promoter (also known asPcor, p6.9 or MP promoter) which is expressed earlier in the baculovirusinfection, and has been shown to be advantageous for expressing secretedproteins (see, for example, Hill-Perkins and Possee, J. Gen. Virol.71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994), andChazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995). In suchtransfer vector constructs, a short or long version of the basic proteinpromoter can be used. Moreover, transfer vectors can be constructedwhich replace the native Zven1/Zven2 secretory signal sequences withsecretory signal sequences derived from insect proteins. For example, asecretory signal sequence from Ecdysteroid Glucosyltransferase (EGT),honey bee Melittin (Invitrogen Corporation; Carlsbad, Calif.), orbaculovirus gp67 (PharMingen: San Diego, Calif.) can be used inconstructs to replace the native Zven1/Zven2 secretory signal sequence.

The recombinant virus or bacmid is used to transfect host cells.Suitable insect host cells include cell lines derived from IPLB-Sf-21, aSpodoptera frugiperda pupal ovarian cell line, such as Sf9 (ATCC CRL1711), Sf21AE, and Sf21 (Invitrogen Corporation; San Diego, Calif.), aswell as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line(Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).Commercially available serum-free media can be used to grow and tomaintain the cells. Suitable media are Sf90 II™ (Life Technologies) orESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRHBiosciences, Lenexa, Kans.) or Express FiveO™ (Life Technologies) forthe T. ni cells. When recombinant virus is used, the cells are typicallygrown up from an inoculation density of approximately 2-5×10⁵ cells to adensity of 1-2×10⁶ cells at which time a recombinant viral stock isadded at a multiplicity of infection (MOI) of 0.1 to 10, more typicallynear 3.

Established techniques for producing recombinant proteins in baculovirussystems are provided by Bailey et al., “Manipulation of BaculovirusVectors,” in Methods in Molecular Biology, Volume 7: Gene Transfer andExpression Protocols, Murray (ed.), pages 147-168 (The Humana Press,Inc. 1991), by Patel et al., “The baculovirus expression system,” in DNACloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages205-244 (Oxford University Press 1995), by Ausubel (1995) at pages 16-37to 16-57, by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995), and by Lucknow, “Insect Cell ExpressionTechnology,” in Protein Engineering: Principles and Practice, Cleland etal. (eds.), pages 183-218 (John Wiley & Sons, Inc. 1996).

Fungal cells, including yeast cells, can also be used to express thegenes described herein. Yeast species of particular interest in thisregard include Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Suitable promoters for expression in yeast includepromoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH(alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinoldehydrogenase), and the like. Many yeast cloning vectors have beendesigned and are readily available. These vectors include YIp-basedvectors, such as YIp5, YRp vectors, such as YRp17, YEp vectors such asYEp13 and YCp vectors, such as YCp19. Methods for transforming S.cerevisiae cells with exogenous DNA and producing recombinantpolypeptides therefrom are disclosed by, for example, Kawasaki, U.S.Pat. No. 4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake,U.S. Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, andMurray et al., U.S. Pat. No. 4,845,075. Transformed cells are selectedby phenotype determined by the selectable marker, commonly drugresistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A suitable vector system for use inSaccharomyces cerevisiae is the POT1 vector system disclosed by Kawasakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Additional suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311, Kingsman etal., U.S. Pat. No. 4,615,974, and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446,5,063,154, 5,139,936, and 4,661,454.

Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii and Candida maltosa are known in the art. See, forexample, Gleeson et al., J. Gen. Microbiol. 132:3459 (1986), and Cregg,U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according tothe methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods fortransforming Acremonium chrysogenum are disclosed by Sumino et al., U.S.Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed byLambowitz, U.S. Pat. No. 4,486,533.

For example, the use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed by Raymond, U.S. Pat. No. 5,716,808,Raymond, U.S. Pat. No. 5,736,383, Raymond et al., Yeast 14:11-23 (1998),and in international publication Nos. WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which can be linearized prior to transformation. Forpolypeptide production in P. methanolica, the promoter and terminator inthe plasmid can be that of a P. methanolica gene, such as a P.methanolica alcohol utilization gene (AUG1 or AUG2). Other usefulpromoters include those of the dihydroxyacetone synthase (DHAS), formatedehydrogenase (FMD), and catalase (CAT) genes. To facilitate integrationof the DNA into the host chromosome, it is preferred to have the entireexpression segment of the plasmid flanked at both ends by host DNAsequences. A suitable selectable marker for use in Pichia methanolica isa P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), andwhich allows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, it is possible to use host cells in which both methanolutilization genes (AUG1 and AUG2) are deleted. For production ofsecreted proteins, host cells can be used that are deficient in vacuolarprotease genes (PEP4 and PRB1). Electroporation is used to facilitatethe introduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. P. methanolica cells can betransformed by electroporation using an exponentially decaying, pulsedelectric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Expression vectors can also be introduced into plant protoplasts, intactplant tissues, or isolated plant cells. Methods for introducingexpression vectors into plant tissue include the direct infection orco-cultivation of plant tissue with Agrobacterium tumefaciens,microprojectile-mediated delivery, DNA injection, electroporation, andthe like. See, for example, Horsch et al., Science 227:1229 (1985),Klein et al., Biotechnology 10:268 (1992), and Miki et al., “Proceduresfor Introducing Foreign DNA into Plants,” in Methods in Plant MolecularBiology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,1993).

Alternatively, Zven genes can be expressed in prokaryotic host cells.Suitable promoters that can be used to express Zven1 or Zven2polypeptides in a prokaryotic host are well-known to those of skill inthe art and include promoters capable of recognizing the T4, T3, Sp6 andT7 polymerases, the P_(R) and P_(L) promoters of bacteriophage lambda,the trp, recA, heat shock, lacUV5, tac, Ipp-lacSpr, phoA, and lacZpromoters of E. coli, promoters of B. subtilis, the promoters of thebacteriophages of Bacillus, Streptomyces promoters, the int promoter ofbacteriophage lambda, the bla promoter of pBR322, and the CAT promoterof the chloramphenicol acetyl transferase gene. Prokaryotic promotershave been reviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson etal., Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), andby Ausubel et al. (1995).

Suitable prokaryotic hosts include E. coli and Bacillus subtilus.Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF′, DH5IMCR, DH10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM10, K38, RR1, Y1088, Y1089,CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), MolecularBiology Labfax (Academic Press 1991)). Suitable strains of Bacillussubtilus include BR151, YB886, MI119, MI120, and B170 (see, for example,Hardy, “Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach,Glover (ed.) (IRL Press 1985)).

When expressing a Zven polypeptide in bacteria such as E. coli, thepolypeptide may be retained in the cytoplasm, typically as insolublegranules, or may be directed to the periplasmic space by a bacterialsecretion sequence. In the former case, the cells are lysed, and thegranules are recovered and denatured using, for example, guanidineisothiocyanate or urea. The denatured polypeptide can then be refoldedand dimerized by diluting the denaturant, such as by dialysis against asolution of urea and a combination of reduced and oxidized glutathione,followed by dialysis against a buffered saline solution. In the lattercase, the polypeptide can be recovered from the periplasmic space in asoluble and functional form by disrupting the cells (by, for example,sonication or osmotic shock) to release the contents of the periplasmicspace and recovering the protein, thereby obviating the need fordenaturation and refolding.

Methods for expressing proteins in prokaryotic hosts are well-known tothose of skill in the art (see, for example, Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995), Ward et al., “Genetic Manipulation andExpression of Antibodies,” in Monoclonal Antibodies: Principles andApplications, page 137 (Wiley-Liss, Inc. 1995), and Georgiou,“Expression of Proteins in Bacteria,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), Chapter 4, starting at page 101(John Wiley & Sons, Inc. 1996), and Rudolph, “Successful Refolding on anIndustrial Scale”, Chapter 10).

Standard methods for introducing expression vectors into bacterial,yeast, insect, and plant cells are provided, for example, by Ausubel(1995).

General methods for expressing and recovering foreign protein producedby a mammalian cell system are provided by, for example, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recoveringprotein produced by a bacterial system is provided by, for example,Grisshammer et al., “Purification of over-produced proteins from E. colicells,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.(eds.), pages 59-92 (Oxford University Press 1995). Established methodsfor isolating recombinant proteins from a baculovirus system aredescribed by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995).

As an alternative, polypeptides of the present invention can besynthesized by exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis. Thesesynthesis methods are well-known to those of skill in the art (see, forexample, Merrifield, J. Am. Chem. Soc. 85:2149 (1963), Stewart et al.,“Solid Phase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co.1984), Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989),Fields and Colowick, “Solid-Phase Peptide Synthesis,” Methods inEnzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al.,Chemical Approaches to the Synthesis of Peptides and Proteins (CRCPress, Inc. 1997)). Variations in total chemical synthesis strategies,such as “native chemical ligation” and “expressed protein ligation” arealso standard (see, for example, Dawson et al., Science 266:776 (1994),Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997), Dawson,Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l Acad. Sci. USA95:6705 (1998), and Severinov and Muir, J. Biol. Chem. 273:16205(1998)).

Peptides and polypeptides of the present invention comprise at leastsix, at least nine, or at least 15 contiguous amino acid residues of SEQID NOs:2 and 5. Illustrative polypeptides of Zven2, for example, include15 contiguous amino acid residues of amino acids 82 to 105 of SEQ IDNO:5. Exemplary polypeptides of Zven1 include 15 contiguous amino acidresidues of amino acids 1 to 32 or amino acids 75 to 108 of SEQ ID NO:2,whereas exemplary Zven2 polypeptides include amino acids 82 to 105 ofSEQ ID NO:5. Within certain embodiments of the invention, thepolypeptides comprise 20, 30, 40, 50, 75, or more contiguous residues ofSEQ ID NOs:2 or 5. Nucleic acid molecules encoding such peptides andpolypeptides are useful as polymerase chain reaction primers and probes.

Examples for the production of Zven1 are shown in Examples 9, 10, 11,and 12.

The present invention contemplates compositions comprising a peptide orpolypeptide described herein. Such compositions can further comprise acarrier. The carrier can be a conventional organic or inorganic carrier.Examples of carriers include water, buffer solution, alcohol, propyleneglycol, macrogol, sesame oil, corn oil, and the like.

7. Isolation of Zven Polypeptides

The polypeptides of the present invention can be purified to at leastabout 80% purity, to at least about 90% purity, to at least about 95%purity, or even greater than 95% purity with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. The polypeptides of the presentinvention can also be purified to a pharmaceutically pure state, whichis greater than 99.9% pure. In certain preparations, a purifiedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin.

Fractionation and/or conventional purification methods can be used toobtain preparations of Zven1 or Zven2 purified from natural sources, andrecombinant Zven polypeptides and fusion Zven polypeptides purified fromrecombinant host cells. In general, ammonium sulfate precipitation andacid or chaotrope extraction may be used for fractionation of samples.Exemplary purification steps may include hydroxyapatite, size exclusion,FPLC and reverse-phase high performance liquid chromatography. Suitablechromatographic media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are preferred. Exemplary chromatographic media include thosemedia derivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties.

Examples of coupling chemistries include cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, hydrazide activation, and carboxyl and amino derivatives forcarbodiimide coupling chemistries. These and other solid media are wellknown and widely used in the art, and are available from commercialsuppliers. Selection of a particular method for polypeptide isolationand purification is a matter of routine design and is determined in partby the properties of the chosen support. See, for example, AffinityChromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988),and Doonan, Protein Purification Protocols (The Humana Press 1996).

Additional variations in Zven isolation and purification can be devisedby those of skill in the art. For example, anti-Zven antibodies,obtained as described below, can be used to isolate large quantities ofprotein by immunoaffinity purification. Moreover, methods for bindingreceptors to ligand polypeptides, such as Zven1 or Zven2, bound tosupport media are well known in the art.

The polypeptides of the present invention can also be isolated byexploitation of particular properties. For example, immobilized metalion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (M.Deutscher, (ed.), Meth. Enzymol. 182:529 (1990)). Within additionalembodiments of the invention, a fusion of the polypeptide of interestand an affinity tag (e.g., maltose-binding protein, an immunoglobulindomain) may be constructed to facilitate purification.

Zven polypeptides or fragments thereof may also be prepared throughchemical synthesis, as described above. Zven polypeptides may bemonomers or multimers; glycosylated or non-glycosylated; pegylated ornon-pegylated; and may or may not include an initial methionine aminoacid residue.

8. Zven Analogs

As described above, the disclosed polypeptides can be used to constructZven variants. These polypeptides can be used to identify Zven1 or Zven2analogs. One type of Zven analog mimics Zven by binding with a Zvenreceptor. Such an analog is considered to be a Zven agonist if thebinding of the analog with a Zven receptor stimulates a response by acell that expresses the receptor. On the other hand, a Zven analog thatbinds with a Zven receptor, but does not stimulate a cellular response,may be a Zven antagonist. Such an antagonist may diminish Zven or Zvenagonist activity, for example, by a competitive or non-competitivebinding of the antagonist to the Zven receptor.

One general class of Zven analogs are agonists or antagonists having anamino acid sequence that has at least one mutation, deletion (amino- orcarboxyl-terminus), or substitution of the amino acid sequencesdisclosed herein. Another general class of Zven analogs is provided byanti-idiotype antibodies, and fragments thereof, as described below.Moreover, recombinant antibodies comprising anti-idiotype variabledomains can be used as analogs (see, for example, Monfardini et al.,Proc. Assoc. Am. Physicians 108:420 (1996)). Since the variable domainsof anti-idiotype Zven antibodies mimic Zven, these domains can provideeither Zven agonist or antagonist activity. As an illustration, Lim andLanger, J. Interferon Res. 13:295 (1993), describe anti-idiotypicinterferon-α antibodies that have the properties of either interferon-αagonists or antagonists.

A third approach to identifying Zven1 or Zven2 analogs is provided bythe use of combinatorial libraries. Methods for constructing andscreening phage display and other combinatorial libraries are provided,for example, by Kay et al., Phage Display of Peptides and Proteins(Academic Press 1996), Verdine, U.S. Pat. No. 5,783,384, Kay, et. al.,U.S. Pat. No. 5,747,334, and Kauffman et al., U.S. Pat. No. 5,723,323.

The activity of a Zven polypeptide, agonist, or antagonist can bedetermined using a standard cell proliferation or differentiation assay.For example, assays measuring proliferation include such assays aschemosensitivity to neutral red dye, incorporation of radiolabelednucleotides, incorporation of 5-bromo-2′-deoxyuridine in the DNA ofproliferating cells, and use of tetrazolium salts (Mosmann, J. Immunol.Methods 65:55 (1983); Porstmann et al., J. Immunol. Methods 82:169(1985); Alley et al., Cancer Res. 48:589 (1988); Cook et al., AnalyticalBiochem. 179:1 (1989); Marshall et al., Growth Reg. 5:69 (1995);Scudiero et al., Cancer Res. 48:4827 (1988); Cavanaugh et al.,Investigational New Drugs 8:347 (1990)). Assays measuringdifferentiation include, for example, measuring cell-surface markersassociated with stage-specific expression of a tissue, enzymaticactivity, functional activity or morphological changes (Raes, Adv. Anim.Cell Biol. Technol. Bioprocesses, pages 161-171 (1989; Watt, FASEB,5:281 (1991); Francis, Differentiation 57:63 (1994)). Assays can be usedto measure other cellular responses, that include, chemotaxis, adhesion,changes in ion channel influx, regulation of second messenger levels andneurotransmitter release. Such assays are well known in the art (see,for example, Chayen and Bitensky, Cytochemical Bioassays: Techniques &Applications (Marcel Dekker 1983)).

The effect of a variant Zven polypeptide, such as Zven1, Zven2, as wellas agonists, fragments, variants and/or chimeras thereof, can also bedetermined by observing contractility of tissues, includinggastrointestinal tissues, with a tensiometer that measures contractilityand relaxation in tissues (see, for example, Dainty et al., J.Pharmacol. 100:767 (1990); Rhee et al., Neurotox. 16:179 (1995);Anderson, Endocrinol. 114:364 (1984); Downing, and Sherwood, Endocrinol.116:1206 (1985)). For example, methods for measuring vasodilatation ofaortic rings are well known in the art. As an illustration, aortic ringsare removed from four-month old Sprague Dawley rats and placed in abuffer solution, such as modified Krebs solution (118.5 mM NaCl, 4.6 mMKCl, 1.2 mM MgSO₄.7H₂O, 1.2 mM KH₂PO₄, 2.5 mM CaCl₂.2H₂O, 24.8 mM NaHCO₃and 10 mM glucose). One of skill in the art would recognize that thismethod can be used with other animals, such as rabbits, other ratstrains, Guinea pigs, and the like. The rings are then attached to anisometric force transducer (Radnoti Inc., Monrovia, Calif.) and the dataare recorded with a Ponemah physiology platform (Gould Instrumentsystems, Inc., Valley View, Ohio) and placed in an oxygenated (95% O₂,5% CO₂) tissue bath containing the buffer solution. The tissues areadjusted to one gram resting tension and allowed to stabilize for aboutone hour before testing. The integrity of the rings can be tested withnorepinepherin (Sigma Co.; St. Louis, Mo.) and carbachol, a muscarinicacetylcholine agonist (Sigma Co.). After integrity is checked, the ringsare washed three times with fresh buffer and allowed to rest for aboutone hour. To test a sample for vasodilatation, or relaxation of theaortic ring tissue, the rings are contracted to two grams tension andallowed to stabilize for fifteen minutes. A Zven polypeptide sample isthen added to one, two, or three of the four baths, without flushing,and tension on the rings recorded and compared to the control ringscontaining buffer only. Enhancement or relaxation of contractility byZven polypeptides, their agonists and antagonists is directly measuredby this method, and it can be applied to other contractile tissues suchas gastrointestinal tissues.

As another example, the effects of Zven1 were tested in a standardguinea pig ileum organ bath. The organ bath system is a standard methodused to measure contractility in isolated tissue, and the guinea pigileum is routinely used for recording contractile responses in theintestine ex vivo (Thomas E., et al., Mol Pharmacol 44:102-10, 1993).Because the components of the enteric nervous system are locatedentirely within the gut, it may be removed from the brain and the spinalcord and its reflex behaviors studied. The classical response observedin gastrointestinal tissue from guinea pig intestinal ileum islongitudinal contraction by smooth muscle fibers orientated along thelong axis of the gut. As shown in Example 6, Zven1 treatment stimulatedsmooth muscle contraction in the ileum at picomolar concentrations aslow as 0.75 ng/ml, which is equivalent to 75 pM. Additionally, thehighest response was observed at the 20 ng/ml zven1 dose. Additionalexamples showing the effects of the Zven molecules of the presentinvention are shown in Examples 7, 14, and 15.

The effect of a variant Zven polypeptide, such as Zven1, Zven2, as wellas agonists, fragments, variants and/or chimeras thereof, on gastricmotility would typically be measured in the clinical setting as the timerequired for gastric emptying and subsequent transit time through thegastrointestinal tract. Gastric emptying scans are well known to thoseskilled in the art, and briefly, comprise use of an oral contrast agent,such as barium, or a radiolabeled meal. Solids and liquids can bemeasured independently. Generally, a test food or liquid is radiolabeledwith an isotope (e.g., ^(99m)Tc), and after ingestion or administration,transit time through the gastrointestinal tract and gastric emptying aremeasured by visualization using gamma cameras (Meyer et al., Am. J. Dig.Dis. 21:296 (1976); Collins et al., Gut 24:1117 (1983); Maughan et al.,Diabet. Med. 13:S6 (1996), and Horowitz et al., Arch. Intern. Med.145:1467 (1985)). The oral administration of phenol red (test meal) tomeasure gastric emptying and intestinal transit in rodents is awell-documented model (Martinez V, Cuttitta F, Tache Y 1997Endocrinology 138:3749-3755). Briefly, animals are deprived of food for18 hours but allowed free access to water. Animals receive oraladministration of 0.15 ml of test meal, consisting of a 1.5% aqueousmethylcellulose solution containing a non-absorbable dye, 0.05% phenolred (50 mg/100 ml Sigma Chemical Company Catalogue # P4758). The effectsof Zven1 on gastric emptying in an in vivo mouse model are shown inExamples 4, 8, 21, and 22. Additional studies can be performed beforeand after the administration of a promotility agent to quantify theefficacy of the Zven polypeptide.

Radiolabeled or affinity labeled Zven polypeptides can also be used toidentify or to localize Zven receptors in a biological sample (see, forexample, Deutscher (ed.), Methods in Enzymol., vol. 182, pages 721-37(Academic Press 1990); Brunner et al., Ann. Rev. Biochem. 62:483 (1993);Fedan et al., Biochem. Pharmacol. 33:1167 (1984)). Also see, Varthakaviand Minocha, J. Gen. Virol. 77:1875 (1996), who describe the use ofanti-idiotype antibodies for receptor identification.

9. Production of Antibodies to Zven Proteins

Antibodies to a Zven polypeptide can be obtained, for example, using theproduct of a Zven expression vector or Zven isolated from a naturalsource as an antigen. Particularly useful anti-Zven1 and anti-Zven2antibodies “bind specifically” with Zven1 and Zven2, respectively.Antibodies are considered to be specifically binding if the antibodiesexhibit at least one of the following two properties: (1) antibodiesbind to Zven1 or Zven2 with a threshold level of binding activity, and(2) antibodies do not significantly cross-react with polypeptidesrelated to Zven1 or Zven2.

With regard to the first characteristic, antibodies specifically bind ifthey bind to a Zven polypeptide, peptide or epitope with a bindingaffinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater,more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ orgreater. The binding affinity of an antibody can be readily determinedby one of ordinary skill in the art, for example, by Scatchard analysis(Scatchard, Ann. NY Acad. Sci. 51:660 (1949)). With regard to the secondcharacteristic, antibodies do not significantly cross-react with relatedpolypeptide molecules, for example, if they detect Zven, but not knownpolypeptides (e.g., known Wnt inhibitors) using a standard Western blotanalysis. Particular anti-Zven1 antibodies bind Zven1, but not Zven2,while certain anti-Zven2 antibodies bind Zven2, but not Zven1.

Anti-Zven1 and anti-Zven2 antibodies can be produced using antigenicZven1 or Zven2 epitope-bearing peptides and polypeptides. Antigenicepitope-bearing peptides and polypeptides of the present inventioncontain a sequence of at least four, or between 15 to about 30 aminoacids contained within SEQ ID NOs:2 or 5. However, peptides orpolypeptides comprising a larger portion of an amino acid sequence ofthe invention, containing from 30 to 50 amino acids, or any length up toand including the entire amino acid sequence of a polypeptide of theinvention, also are useful for inducing antibodies that bind with Zven1or Zven2. It is desirable that the amino acid sequence of theepitope-bearing peptide is selected to provide substantial solubility inaqueous solvents (i.e., the sequence includes relatively hydrophilicresidues, while hydrophobic residues are preferably avoided). Moreover,amino acid sequences containing proline residues may be also bedesirable for antibody production.

As an illustration, potential antigenic sites in Zven1 or Zven2 wereidentified using the Jameson-Wolf method, Jameson and Wolf, CABIOS4:181, (1988), as implemented by the PROTEAN program (version 3.14) ofLASERGENE (DNASTAR; Madison, Wis.). Default parameters were used in thisanalysis.

The Jameson-Wolf method predicts potential antigenic determinants bycombining six major subroutines for protein structural prediction.Briefly, the Hopp-Woods method, Hopp et al., Proc. Nat'l Acad. Sci. USA78:3824 (1981), was first used to identify amino acid sequencesrepresenting areas of greatest local hydrophilicity (parameter: sevenresidues averaged). In the second step, Emini's method, Emini et al., J.Virology 55:836 (1985), was used to calculate surface probabilities(parameter: surface decision threshold (0.6)=1). Third, theKarplus-Schultz method, Karplus and Schultz, Naturwissenschaften 72:212(1985), was used to predict backbone chain flexibility (parameter:flexibility threshold (0.2)=1). In the fourth and fifth steps of theanalysis, secondary structure predictions were applied to the data usingthe methods of Chou-Fasman, Chou, “Prediction of Protein StructuralClasses from Amino Acid Composition,” in Prediction of Protein Structureand the Principles of Protein Conformation, Fasman (ed.), pages 549-586(Plenum Press 1990), and Garnier-Robson, Garnier et al., J. Mol. Biol.120:97 (1978) (Chou-Fasman parameters: conformation table=64 proteins; αregion threshold=103; β region threshold=105; Garnier-Robson parameters:α and β decision constants=0). In the sixth subroutine, flexibilityparameters and hydropathy/solvent accessibility factors were combined todetermine a surface contour value, designated as the “antigenic index.”Finally, a peak broadening function was applied to the antigenic index,which broadens major surface peaks by adding 20, 40, 60, or 80% of therespective peak value to account for additional free energy derived fromthe mobility of surface regions relative to interior regions. Thiscalculation was not applied, however, to any major peak that resides ina helical region, since helical regions tend to be less flexible.

The results of this analysis indicated that suitable antigenic peptidesof Zven1 include the following segments of the amino acid sequence ofSEQ ID NO:2: amino acids 22 to 27 (“antigenic peptide 1”), amino acids33 to 41 (“antigenic peptide 2”), amino acids 61 to 68 (“antigenicpeptide 3”), amino acids 80 to 85 (“antigenic peptide 4”), amino acids97 to 102 (“antigenic peptide 5”), and amino acids 61 to 85 (“antigenicpeptide 6”). The present invention contemplates the use of any one ofantigenic peptides 1 to 6 to generate antibodies to Zven1. The presentinvention also contemplates polypeptides comprising at least one ofantigenic peptides 1 to 6.

Similarly, analysis of the Zven2 amino acid sequence indicated thatsuitable antigenic peptides of Zven2 include the following segments ofthe amino acid sequence of SEQ ID NO:5: amino acids 25 to 33 (“antigenicpeptide 7”), amino acids 53 to 66 (“antigenic peptide 8”), amino acids88 to 95 (“antigenic peptide 9”), amino acids 98 to 103 (“antigenicpeptide 10”), and amino acids 88 to 103 (“antigenic peptide 11”). Thepresent invention contemplates the use of any one of antigenic peptides7 to 11 to generate antibodies to Zven2. The present invention alsocontemplates polypeptides comprising at least one of antigenic peptides7 to 11.

Polyclonal antibodies to recombinant Zven protein or to Zven isolatedfrom natural sources can be prepared using methods well-known to thoseof skill in the art. See, for example, Green et al., “Production ofPolyclonal Antisera,” in Immunochemical Protocols (Manson, ed.), pages1-5 (Humana Press 1992), and Williams et al., “Expression of foreignproteins in E. coli using plasmid vectors and purification of specificpolyclonal antibodies,” in DNA Cloning 2: Expression Systems, 2ndEdition, Glover et al. (eds.), page 15 (Oxford University Press 1995).The immunogenicity of a Zven polypeptide can be increased through theuse of an adjuvant, such as alum (aluminum hydroxide) or Freund'scomplete or incomplete adjuvant. Polypeptides useful for immunizationalso include fusion polypeptides, such as fusions of Zven or a portionthereof with an immunoglobulin polypeptide or with maltose bindingprotein. The polypeptide immunogen may be a full-length molecule or aportion thereof. If the polypeptide portion is “hapten-like,” suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

Although polyclonal antibodies are typically raised in animals such ashorses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, orsheep, an anti-Zven antibody of the present invention may also bederived from a subhuman primate antibody. General techniques for raisingdiagnostically and therapeutically useful antibodies in baboons may befound, for example, in Goldenberg et al., international patentpublication No. WO 91/11465, and in Losman et al., Int. J. Cancer 46:310(1990).

Alternatively, monoclonal anti-Zven antibodies can be generated. Rodentmono-clonal antibodies to specific antigens may be obtained by methodsknown to those skilled in the art (see, for example, Kohler et al.,Nature 256:495 (1975), Coligan et al. (eds.), Current Protocols inImmunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991)[“Coligan”], Picksley et al., “Production of monoclonal antibodiesagainst proteins expressed in E. coli,” in DNA Cloning 2: ExpressionSystems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford UniversityPress 1995)).

Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising a Zven gene product, verifying the presence ofantibody production by removing a serum sample, removing the spleen toobtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive cloneswhich produce antibodies to the antigen, culturing the clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

In addition, an anti-Zven antibody of the present invention may bederived from a human monoclonal antibody. Human monoclonal antibodiesare obtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge. In thistechnique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described, for example, by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994).

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).

For particular uses, it may be desirable to prepare fragments ofanti-Zven antibodies. Such antibody fragments can be obtained, forexample, by proteolytic hydrolysis of the antibody. Antibody fragmentscan be obtained by pepsin or papain digestion of whole antibodies byconventional methods. As an illustration, antibody fragments can beproduced by enzymatic cleavage of antibodies with pepsin to provide a 5Sfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent to produce 3.5S Fab′ monovalent fragments.Optionally, the cleavage reaction can be performed using a blockinggroup for the sulfhydryl groups that result from cleavage of disulfidelinkages. As an alternative, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. No. 4,331,647,Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960), Porter, Biochem.J. 73:119 (1959), Edelman et al., in Methods in Enzymology Vol. 1, page422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described by Inbar etal., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde (see, for example,Sandhu, Crit. Rev. Biotech. 12:437 (1992)).

The Fv fragments may comprise V_(H) and V_(L) chains, which areconnected by a peptide linker. These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains which areconnected by an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cell,such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are described, for example, by Whitlow etal., Methods: A Companion to Methods in Enzymology 2:97 (1991) (alsosee, Bird et al., Science 242:423 (1988), Ladner et al., U.S. Pat. No.4,946,778, Pack et al., Bio/Technology 11:1271 (1993), and Sandhu,supra).

As an illustration, a scFV can be obtained by exposing lymphocytes toZven polypeptide in vitro, and selecting antibody display libraries inphage or similar vectors (for instance, through use of immobilized orlabeled Zven protein or peptide). Genes encoding polypeptides havingpotential Zven polypeptide binding domains can be obtained by screeningrandom peptide libraries displayed on phage (phage display) or onbacteria, such as E. coli. Nucleotide sequences encoding thepolypeptides can be obtained in a number of ways, such as through randommutagenesis and random polynucleotide synthesis. These random peptidedisplay libraries can be used to screen for peptides, which interactwith a known target that can be a protein or polypeptide, such as aligand or receptor, a biological or synthetic macromolecule, or organicor inorganic substances. Techniques for creating and screening suchrandom peptide display libraries are known in the art (Ladner et al.,U.S. Pat. No. 5,223,409, Ladner et al., U.S. Pat. No. 4,946,778, Ladneret al., U.S. Pat. No. 5,403,484, Ladner et al., U.S. Pat. No. 5,571,698,and Kay et al., Phage Display of Peptides and Proteins (Academic Press,Inc. 1996)) and random peptide display libraries and kits for screeningsuch libraries are available commercially, for instance from CLONTECHLaboratories, Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego,Calif.), New England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKBBiotechnology Inc. (Piscataway, N.J.). Random peptide display librariescan be screened using the Zven sequences disclosed herein to identifyproteins which bind to Zven.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106 (1991),Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166 (Cambridge University Press 1995), andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137 (Wiley-Liss, Inc. 1995)).

Alternatively, an anti-Zven antibody may be derived from a “humanized”monoclonal antibody. Humanized monoclonal antibodies are produced bytransferring mouse complementary determining regions from heavy andlight variable chains of the mouse immunoglobulin into a human variabledomain. Typical residues of human antibodies are then substituted in theframework regions of the murine counterparts. The use of antibodycomponents derived from humanized monoclonal antibodies obviatespotential problems associated with the immunogenicity of murine constantregions. General techniques for cloning murine immunoglobulin variabledomains are described, for example, by Orlandi et al., Proc. Nat'l Acad.Sci. USA 86:3833 (1989). Techniques for producing humanized monoclonalantibodies are described, for example, by Jones et al., Nature 321:522(1986), Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285 (1992),Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer et al., J. Immun.150:2844 (1993), Sudhir (ed.), Antibody Engineering Protocols (HumanaPress, Inc. 1995), Kelley, “Engineering Therapeutic Antibodies,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 399-434 (John Wiley & Sons, Inc. 1996), and by Queen et al., U.S.Pat. No. 5,693,762 (1997).

Polyclonal anti-idiotype antibodies can be prepared by immunizinganimals with anti-Zven antibodies or antibody fragments, using standardtechniques. See, for example, Green et al., “Production of PolyclonalAntisera,” in Methods In Molecular Biology: Immunochemical Protocols,Manson (ed.), pages 1-12 (Humana Press 1992). Also, see Coligan at pages2.4.1-2.4.7. Alternatively, monoclonal anti-idiotype antibodies can beprepared using anti-Zven antibodies or antibody fragments as immunogenswith the techniques, described above. As another alternative, humanizedanti-idiotype antibodies or subhuman primate anti-idiotype antibodiescan be prepared using the above-described techniques. Methods forproducing anti-idiotype antibodies are described, for example, by Irie,U.S. Pat. No. 5,208,146, Greene, et. al., U.S. Pat. No. 5,637,677, andVarthakavi and Minocha, J. Gen. Virol. 77:1875 (1996).

10. Therapeutic Uses of Zven Polypeptides

The present invention includes the use of proteins, polypeptides, andpeptides having Zven activity (such as Zven polypeptides, Zven analogs,active Zven anti-idiotype antibodies, and Zven fusion proteins) to asubject, which lacks an adequate amount of this polypeptide. The presentinvention contemplates both veterinary and human therapeutic uses.Illustrative subjects include mammalian subjects, such as farm animals,domestic animals, and human patients.

Zven polypeptides, such as Zven1, Zven2, as well as agonists, fragments,variants and/or chimeras thereof, and antagonists thereof are useful indiseases characterized by dysfunction of the gastrointestinal tract dueto limited contractility, gastric emptying, and/or increasedcontractility, as well as disorders associated with inflammation of theintestine.

Dysfunction of the gastrointestinal tract due to limited contractilityand/or gastric emptying is a characteristic of diseases and disordersincluding, but not limited to, post-operative ileus, post-partum ileus,chronic constipation, dyspepsia, intestinal pseudo-obstruction,gastroparesis, diabetic gastroparesis, gastroesophageal reflux, emesis,use or consumption of opiods and/or narcotics, muscular dystrophy,prgressive systemic sclerosis, infectious diarrhea, and paralyticgastroparesis.

Postoperative inhibition of gastrointestinal motility (postoperativeileus) is induced by laparotomy and intra-abdominal procedures. Thetransient inhibition of gastrointestinal motility occurring in humansmainly in the stomach and the colon may last for several days and canconsiderably contribute to a patient's postoperative discomfort. Oralfood intake may be delayed until post operative ileus has been resolved,and prolonged nasogastric suction or, in rare cases, even relaparatomybecomes necessary (Livingston, E. et al., Post operataive ileu. Dig. DisSci 35:121-132, 1990). Zven1 is a potent stimulator of gastrointestinalcontractility as shown in the following examples. As such, Zvenpolypeptides, such as Zven1, Zven2, as well as agonists, fragments,variants and/or chimeras thereof, can be used to stimulategastrointestinal contractility in patients after surgery. For example,patients who have disorders such as, post-surgical gastroparesis,including post-operative ileus, are good candidates for administrationof Zven polypeptides, such as Zven1, Zven2, as well as agonists,fragments, variants and/or chimeras thereof.

Post-operative ileus (POI) is a condition of reduced intestinal tractmotility, including delayed gastric emptying, that occurs as a result ofdisrupted muscle tone following surgery. It is especially problematicfollowing abdominal surgery. The problem may arise from the surgeryitself, from the residual effects of anesthetic agents, andparticularly, from pain-relieving narcotic and opiate drugs used duringand after surgery.

Post-operative ileus reduces gastrointestinal motility, which also maydelay the absorption of drugs administered orally. Reduced intestinalmotility following surgery is a major cause of extended hospital stays,which are extremely expensive and result in an increased chance ofdeveloping other complications. Extended durations of POI may requirethe use of parenteral nutrition, which also is expensive. With theincreasing cost of medical care, the expenses associated with hospitalstays and parenteral feeding are expected to increase even further.

The acute nature of this condition provides an opportunity to treat withZven polypeptides, such as Zven1, Zven2, as well as agonists, fragments,variants and/or chimeras thereof. Moreover, since oral drugs would becounter-indicated during POI, a drug administered subcutaneously,intramuscularly, or intravenously, such as Zven1 would be beneficial.“Prokinetics” have been found to alleviate the symptoms associated withPOI, and since Zven1 has prokinetic properties, can be effective intreating POI. Currently there are very few drugs that can effectivelytreat POI, and those that are available have side effects, cannot betaken with other medications, and/or are administered orally.

The effect of a Zven polypeptide, such as Zven1, Zven2, as well asagonists, fragments, variants and/or chimeras thereof, on POI can bemeasured in an in vivo model by administering it orally (p.o.),intraperitoneally (i.p.), intraveneously (i.v.), subcutaneously (s.c.),or intramuscularly (i.m.) to fasted animals at an appropriate time priorto or following a laparotomy and cecal manipulation performed underanesthesia. One of the Major Models listed below may then be used toassess extent of gastric emptying and/or intestinal transit at timesranging from 10 to 180 minutes after removal of the anesthetic. In dogmodels, this time may be greater (up to 50 h). Using severalpost-surgical time points allows an estimate of the effects of surgeryon gastric emptying and transit along much of the gastrointestinaltract. This model has been used extensively to evaluate the efficacy ofprokinetic drugs on gastric emptying and/or intestinal transit as aresult of POI (e.g. Martinez, Rivier, and Tache. J. Pharmacol.Experimental. Therap. 290:629 (1999) and Furuta et al. Biol. Pharm.Bull. 25:103-1071 (2002)).

Additionally, Zven polypeptides, such as Zven1, Zven2, as well asagonists, fragments, variants and/or chimeras thereof, can be used toprevent POI. In this scenario, the Zven polypeptides, such as Zven1,Zven2, as well as agonists, fragments, variants and/or chimeras thereof,are administered to the patient pre-operatively.

Diabetic gastroparesis is paralysis of the stomach brought about by amotor abnormality in the stomach, as a complication of both type I andtype II diabetes. It is characterized by delayed gastric emptying,post-prandial distention, nausea and vomiting. In diabetes, it isthought to be due to a neuropathy, though it is also associated withloss of interstitial cells of Cajal (ICC), which are the “pacemakercells” of the gut.

Patients who have diabetes mellitus may also have disorders related togastric emptying. For example, a patient who has had diabetes mellitusfor at least five years may have a prevalence of significant delay ingastric emptying of >50% (Horowitz, M, et al., J. GastoenterologyHepatology: 1:97-113, 1986). Gastric neuromuscular dysfunction occurs inup to 30-50% of patients after 10 years of type 1 or type 2 diabetes(Koch K., et al., Dig Dis Sci 44:1061-1075, 1999). Zven1, Zven2, and/ortheir agonists may also be used as treatment for diabetic patients.

The often-acute nature of the episodes of diabetic gastroparesisprovides an opportunity to treat with Zven polypeptides, such as Zven1,Zven2, as well as agonists, fragments, variants and/or chimeras thereof.“Prokinetics” have been found to alleviate the delayed gastric emptyingassociated with diabetic gastroparesis. Currently there are very fewdrugs that can effectively treat diabetic gastroparesis, and those thatare available have side effects and/or cannot be taken with othermedications. Oral drugs may not be tolerated during severe episodes, andthus, would require intravenous administration of a prokinetic.

The spontaneously diabetic NOD/LtJ mouse (available from JacksonLaboratories) develops delayed gastric emptying, impaired electricalpacemaking, and reduced motor neurotransmission. This is described inWatkins et al. J. Clin. Invest. 106:373-384 (2000). This strain alsoappears to have defects in interstitial cells of Cajal (ICC) networksthat are associated with impaired motility. Streptozotocin treatment ofrats and mice is a well-recognized and acceptable method to inducediabetes; these animals are characterized by impaired gastric emptyingand intestinal transit, and thus, show symptoms of diabeticgastroparesis (Yamano et al. Naunyn-Schmiedeberg's Arch. Pharmacol. 356:145-150 (1997) and Watkins et al. J. Clin. Invest. 106:373-384 (2000)).The ability of Zven polypeptide, such as Zven1, Zven2, as well asagonists, fragments, variants and/or chimeras thereof, (administered viap.o., i.v., i.p., s.c., or i.m.) to improve the impaired gastricemptying and intestinal transit associated with the diabetes, can alsobe measured by one of the Major Models described below.

Inflammatory reactions cause various clinical manifestations frequentlyassociated with abnormal motility of the gastrointestinal tract, such asnausea, vomiting, ileus or diarrhea. Bacterial lipopolysaccharide (LPS)exposure, for example, induces such an inflammatory condition, which isobserved in both humans and experimental animals, and is characterizedby biphasic changes in gastrointestinal motility: increased transit inearlier phases and delayed transit in later phases. Since Zven1 plays arole in inflammation, and has biphasic activities at low (prokinetic)and high (inhibitory) doses, it will be beneficial in these inflammatoryconditions.

Zven polypeptides, such as Zven1, Zven2, as well as agonists, fragments,variants and/or chimeras thereof, can also be used to treatgastrointestinal related sepsis. Experimental “sepsis”/endotoxemia isproduced in rodents using methods described in Ceregrzyn et al.Neurogastroenterol. Mot. 13:605-613 (2001). These animals developbiphasic alterations in gastrointestinal transit. A Zven polypeptide,such as Zven1, Zven2, as well as agonists, fragments, variants and/orchimeras thereof, can be administered (via p.o., i.v., i.p., s.c., ori.m.) at either low (prokinetic) or high (inhibitory) concentrations,depending on the phase of the disease. Gastric emptying and/orintestinal transit would then be measured using one of the Major Modelsdescribed below.

Morphine and other opioid analgesics are some of the most common painrelievers used, especially following surgery. Because they inhibit therelease of acetylcholine from the mesenteric plexus and thereby reducethe propulsive activity in the gastrointestinal tract, individualstaking opioid analgesics often suffer from reduced gastric emptying andintestinal transit. Since Zven1 simulates intestinal contractility andhas gastrointestinal prokinetic activity, Zven1 may be beneficial in thetreatment of opioid-induced motility disorder(s).

The effect of Zven1 on opioid-induced gastroparesis in experimentalrodents can be measured by a well-known model. See Suchitra et al. WorldJ. Gastroenterol. 9:779-783 (2003) and Asai, Arzneim.-Forsch./Drug Res.48:802-805 (1998). Mice or rats are administered a drug from the opioidclass (e.g. morphine) via the appropriate route of administration (p.o.,i.p., i.v., s.c., i.m.) at a dose known to inhibit gastric emptying andintestinal transit (e.g. 1-5 mg/kg BW) at a set time prior toadministration of the test agent or method used to monitor gastricemptying and intestinal transit (by use of one of the Major Modelslisted below). The Zven polypeptide, such as Zven1, Zven2, as well asagonists, fragments, variants and/or chimeras thereof, is administeredat the appropriate time point (via p.o., i.v., i.p., s.c., or i.m.) inrelation to the opioid and test meal or method to assess its efficacy inrelieving opioid-induced gastroparesis/ileus.

Individuals with neuropathies (e.g. as seen with diabetes) often sufferfrom gastroparesis and reduced intestinal motility, as a result of amalfunctioning nervous system. Since Zven1 appears to induce intestinalcontractility independently from nervous input, this would suggest thatZven1 would be beneficial in individuals suffering fromneuropathy-induced gastrointestinal disorders.

The effect of Zven1 on vagotomy-induced gastroparesis in experimentalmammals can be measured in an animal model. Thoracic vagotomy isperformed in experimental mammals as described, for example, in Takedaet al. Jpn. J. Pharmacol. 81:292-297 (1999) and Hatanaka et alNeurogastroenterol. Motil. 8:227-233 (1996). These animals arecharacterized by reduced gastric emptying and intestinal transit. TheZven polypeptide, such as Zven1, Zven2, as well as agonists, fragments,variants and/or chimeras thereof, is administered (via p.o., i.p., i.v.,s.c., or i.m.) as a means to alleviate this vagotomy-inducedgastroparesis/ileus, which is monitored using one of the Major Modelslisted below.

Since Zven1 has contractile activity in the intestinal organ bath systemin the presence of atropine, Zven1 can also have intestinal activity invivo in the presence of atropine. Atropine is a compound that blockscholinergic nerve potential. Therefore, the ability of Zven1 to beactive in the presence of atropine suggests that Zven1 can actindependent of the cholinergic nervous system, and thus, can be abeneficial treatment to those suffering from neuropathy-associatedgastroparesis and/or ileus.

Another indication where Zven1 and/or Zven2 can be used to treat gastricdysfunction is gastreoesophageal reflux disease, which is characterizedby the backward flow of the stomach contents into the esophagus, oftenas a result of a reduction in the pressure barrier due to the failure ofthe lower esophageal sphincter. Prokinetics, such as bethanechol(Urecholine) and metoclopramide (Reglan) have been shown to helpstrengthen the sphincter and make the stomach empty faster.Metoclopramide also improves muscle action in the digestive tract, butthese drugs have frequent side effects that limit their usefulness. Thusa biologic prokinetic, such as a Zven polypeptide, including Zven1,Zven2, as well as agonists, fragments, variants and/or chimeras thereof,that improves contraction in the stomach and gastrointestinal tract,with or without improved stability of the esophageal sphincter will beuseful to treat gastroesophageal reflux disease.

Methods to investigate effects of atropine in vivo in experimentalrodents are well known in the art. See Chadhuri et al. Life Sciences66:847-854 (2000) and Kaneko et al. Digest. Dis. Sci. 40:2043-2051(1995). Mice or rats are administered atropine via the appropriate routeof administration (p.o., i.p., i.v., s.c., i.m.) at a dose known toinhibit gastric emptying and intestinal transit (e.g. 0.1-2.0 mg/kg BW)at a set time prior to administration of the test agent or method usedto monitor gastric emptying and intestinal transit (by use of one of theMajor Models listed below). Zven polypeptides, such as Zven1, Zven2, aswell as agonists, fragments, variants and/or chimeras thereof, isadministered at the appropriate time point (via p.o., i.v., i.p., s.c.,or i.m.) in relation to the atropine and test meal or method to assessits efficacy in the presence of atropine.

Additional indications where Zven polypeptides, such as Zven1, Zven2, aswell as agonists, fragments, variants and/or chimeras thereof, can beused to treat gastric dysfunction are gastroparesis, a paralysis of thestomach brought about by a motor abnormality in the stomach or as acomplication of diseases such as diabetes, progressive systemicsclerosis, anorexia nervosa or myotonic dystrophy. Diabeticgastroparesis results in delayed gastric emptying, followed bypost-prandial distention and vomiting, which can result in poor glycemiccontrol. It is often associated with loss of interstitial cells of Cajal(ICC). Zven polypeptides, such as Zven1, Zven2, as well as agonists,fragments, variants and/or chimeras thereof, can also be used to treatgastric dysfunction observed or associated with chronic constipationwhich can be characterized by intestinal hypomotility, often due to lackof intestinal muscle tone or intestinal spasticity. Another indicationwhere Zven polypeptides, such as Zven1, Zven2, as well as agonists,fragments, variants and/or chimeras thereof, can be used to treatgastric dysfunction is dyspepsia, which is defined as an impairment ofthe power or function of digestion. It can be a symptom of a primarygastrointestinal dysfunction, or a complication of appendicitis, gallbladder disease or malnutrition. Zven polypeptides, such as Zven1,Zven2, as well as agonists, fragments, variants and/or chimeras thereof,can also be used to treat gastric dysfunction from emesis which ischaracterized by symptoms of nausea and vomiting, induced spontaneously,as a result of delayed gastric emptying, or associated with emetogeniccancer chemotherapy or irradiation therapy. In still another indicationwhere Zven polypeptides, such as Zven1, Zven2, as well as agonists,fragments, variants and/or chimeras thereof, can be used to treatgastric dysfunction associated with paralytic gastroparesis. This is aparalysis of the stomach brought about by a motor abnormality in thestomach or as a complication of diseases (other than diabetes) such asprogressive systemic sclerosis, anorexia nervosa or myotonic dystrophy.It results in delayed gastric emptying, followed by post-prandialdistention and vomiting.

For disorders related to deficient gastrointestinal function, clinicalsigns of improved function include, but are not limited to, increasedintestinal transit, increased gastric emptying, flatus, and borborygmi,ability to consume liquids and solids, and/or a reduction in nauseaand/or emesis.

Additionally, since Zven1 and Zven2 reduce contractility whenadministered at high doses, Zven polypeptides, such as Zven1, Zven2, aswell as agonists, fragments, variants and/or chimeras thereof, can beused to reduce or inhibit contractility when such effect is desired.This effect may be desired as a solo therapy to treat, for example,diarrhea, including chronic diarrhea and traveler's diarrhea.

For disorders related to hyperactive gastrointestinal contractility,clinincal signs of improved gastrointestinal function include, but arenot limited to, slowed gastric emptying, slowed intestinal transit,and/or a reduction in cramps associated with diarrhea.

Zven polypeptides, such as Zven1, Zven2, as well as agonists, fragments,variants and/or chimeras thereof, can be used to stimulate chemokineproduction. Chemokines are small pro-inflammatory proteins that have abroad range of activities involved in the recruitment and function ofleukocytes. Rat CINC-1, murine KC, and human GROα are members of the CXCsubfamily of chemokines. Chemokines, in general, can be divided intogroups that are chemotactic predominatly for neutrophils, and also haveangiogenic activity, and those that primarily attract T lymphocytes andmonocytes. See Banks, C. et al, J. Pathology 199: 28-35, 2002.Chemokines in the first group display an ELR (Glu-Leu-Arg) amino acidmotif at the NH₂ terminus. GROα, for example, contains this motif. GROαalso has mitogenic and angiogenic properties and is involved in woundhealing and blood vessel formation. (See, for example, Li and Thomhill,Cytokine 12:1409 (2000)). As illustrated by Examples 2, 3, and 17, Zven1and Zven2 stimulated the release of chemokine CINC-1 (Cytokine InducedNeutrophil Chemoattractant factor 1) in cell lines derived from thethoracic aorta of rats, Zven1 stimulated the release of chemokine KCfrom mice, and chemokine MIP-2 (mouse Macrophage Inflammatory Protein-2)is up-regulated in response to a low dose (intraperitoneal injection) ofZven1. Therefore, Zven polypeptides, such as Zven1, Zven2, as well asagonists, fragments, variants and/or chimeras thereof, can be used tostimulate the production chemokines in vivo. The chemokines can bepurified from culture media and used in research or clinical settings.Zven variants can also be identified by the ability to stimulateproduction of chemokines in vitro or in vivo.

Upregulated chemokine expression correlates with increasing activity ofIBD. See Banks, C. et al, J. Pathology 199: 28-35, 2002. Chemokines areable to attract inflammatory cells and are involved in their activation.Similarly, MIP-2 expression has been found to be associated withneutrophil influx in various inflammatory conditions. As polypeptidesthat stimulate the production of chemokines, Zven polypeptides, such asZven1, Zven2, as well as agonists, fragments, variants and/or chimerasthereof, may be useful in treating Inflammatory Bowel Disease byreducing, inhibiting or preventing chemokine influx in the intestinaltract.

Similarly, as shown in Example 3, Zven1 administration can causeneutrophil infiltration. There are many aspects involved in the immuneresponse of a mammal to an injury or infection where neutrophilinfiltration would be desirable. As such, Zven polypeptides, such asZven1, Zven2, as well as agonists, fragments, variants and/or chimerasthereof, will be useful as an agent to induce neutrophil infiltration.

As a protein that can stimulate the production of chemokines, Zvenpolypeptides, such as Zven1, Zven2, as well as agonists, fragments,variants and/or chimeras thereof, may be useful in treating infections,including fungal, bacterial, viral and parasitic infections. Thus, theadministration of a Zven polypeptide, such as Zven1, Zven2, as well asan agonist, fragment, variant and/or a chimera thereof, may be used asan immune booster to a specific tissue site. For example, Zven1administered to gastrointestinal tissue, or to lung tissue, may beuseful alone, or in combination therapy to treat infections.

Example 5 demonstrates that Zven1 and Zven2 can stimulate angiogenesis.Accordingly, Zven1, Zven2, Zven1 agonists, and Zven2 agonists can beused to stimulate proliferation of cardiac stem cells. These moleculescan be administered alone or in combination with other angiogenicfactors, such as vascular endothelial growth factor.

11. Major Models Used to Measure Gastric Emptying and Intestinal Transit

As described above, there are a number of in vivo models to measuregastric function. A few of these models are represented below.

Model 1: Method to Measure Rate and Extent of Gastric Emptying andIntestinal Transit using Phenol Red/Methyl Cellulose in ExperimentalMammals

Fasted animals are given Zven1 (or other Zven agent, Zven polypeptides,such as Zven1, Zven2, as well as agonists, fragments, variants and/orchimeras thereof) by the appropriate route (p.o., i.p., i.v., s.c.,i.m.). At the appropriate time point, a non-nutritive semi-solid mealconsisting of methylcellulose and phenol red is administered by gavage,and animals are sacrificed at a set time following this mealadministration. Transit is assessed by the recovery andspectrophotometric determination of phenol red from designated regionsalong the gastrointestinal tract. The period of dye recovery in thegastrointestinal tract may be from 10 to 180 minutes, depending on theindication and intestinal site of interest. This model has been usedextensively to evaluate the efficacy of other prokinetic drugs ongastric emptying and/or intestinal transit.

Model 2: Method to Measure Rate and Extent of Intestinal Transit usingArabic Gum/Charcoal Meal in Experimental Mammals

Fasted animals would be given Zven1 (or other Zven agent, Zvenpolypeptides, such as Zven1, Zven2, as well as agonists, fragments,variants and/or chimeras thereof) by the appropriate route (i.p., i.v.,s.c., i.m., p.o.). At the appropriate time point, a semi-solid mealconsisting of gum arabic and charcoal is administered by gavage, andanimals are sacrificed at a set time following this meal administration(Puig and Pol. J. Pharmacol. Experiment. Therap. 287:1068 (1998)).Transit is assessed by the distance that the charcoal meal traveled as afraction of the total distance of the intestine. The period of transitmeasurement in the gastrointestinal tract may be from 10 to 180 minutes,depending on the indication and intestinal site of interest. This modelhas been used extensively to evaluate the efficacy of prokinetic drugson intestinal transit.

Model 3: Method to Measure Rate and Extent of Gastric Emptying usingPolystyrene Beads (Undigestible Solids) in Experimental Rodents

Gastric emptying is evaluated by determining the emptying of polystyrenebeads of a specific diameter (e.g. 1 mm for rats) from the stomach offasted (24 h) male or female experimental rodents in response to Zven1(or another Zven agent, Zven polypeptides, such as Zven1, Zven2, as wellas agonists, fragments, variants and/or chimeras thereof) via p.o.,i.p., s.c., i.v. or i.m. route of administration. Polystyrene beads areadministered by gavage and assessed for emptying as previously described(Takeuchi et al. Digest. Dis. Sci. 42; 251-258 (1997)). Animals aresacrificed at a specified time after pellet administration (e.g. 20-180min), and the stomachs are removed. The number of the pellets remainingin the stomach are counted. In control studies, 90% of pellets would beexpected to remain in the stomach after 30 min, and fewer than 10% inthe stomach after 3 h. This model has been used extensively to evaluatethe gastric emptying efficacy of prokinetic drugs in experimentalrodents.

Model 4: Method to Measure Rate and Extent of Gastric Emptying of aLiquid or Solid Test Meal in Experimental Mammals using Acetaminophen asthe Tracer

Fasted animals are given a liquid or solid test meal containingacetominophen as the tracer. The test compound (e.g. Zven1) isadministered p.o., i.v., i.p., s.c., or i.m. either before or after testmeal administration. Blood samples are obtained at intervals between 0and 120 min, and the plasma concentration of acetaminophen (which is ameasure of gastric emptying) is measured by HPLC. This is described, forexample, in Trudel et al Peptides 24:531-534 (2003).

Model 5: Method to Measure Gastric Emptying of a Solid Meal inExperimental Rodents

Mice or rats (“rodents”) are separated into four groups (Zven1-;positive control-[erythromycin, metoclopramide, or cisapride]; negativecontrol-[caerulein]; and vehicle-treated groups). Each group containsapproximately 10 animals. They are deprived of food for 24 hours, buthave free access to water during fast period. Animals are housed one percage, with floor grids placed in the cages to prevent contact with thebedding or feces. The fasted animals are treated with one of the aboveagents via one of the following routes of administrations: oral; i.p.,i.v., s.c., or i.m.). Animals are introduced to pre-weighed Purina chowindividually for a set period of time (e.g. 1 hr) in their home cages(with bedding removed) at the appropriate time point following or priorto administration of test agent. At the end of the feeding period,animals are housed in their home cages without food and water for anadditional set period of time. They are then euthanized, the abdominalcavity opened, and stomach removed after clamping the pylorus andcardia. The stomach is weighed, opened, and washed of the gastriccontent by tap water. The gastric wall is wiped dry, and the emptystomach is weighed again. Gastric contents are collected, dried, andweighed. The amount of food contained in the stomach (as measured ingrams) is calculated as the difference between the total weight of thestomach with content and the weight of the stomach wall after thecontents are removed. The weight of the pellet and spill in the cage isalso measured at the end of the feeding period. The solid food ingestedby the animals is determined by the difference between the weight of thePurina chow before feeding and the weight of the pellet and spill at theend of the feeding period. The gastric emptying for the designatedperiod is calculated according to the equation: % of gastricemptying=(1−gastric content/food intake)×100. This model has been usedextensively in the literature to assess gastric emptying of a solid meal(Martinez et al. J. Pharmacol. Experiment. Ther. 301: 611-617 (2002)).

Model 6: Method to Measure Rate and Extent of Gastric Emptying of aSolid Test Meal in Experimental Mammals

Fasted animals receive barium sulfate spheroids with a standard meal,followed by administration of the test compound such as Zvenpolypeptides, such as Zven1, Zven2, as well as agonists, fragments,variants and/or chimeras thereof (via p.o., i.v., i.p., s.c., or i.m)either before or after the meal. Gastric emptying is measured by meansof X-ray location, with passage being monitored at least every 15 min-2h. This method is described, for example, in Takeda et al. Jpn. J.Pharmacol. 81:292-297 (1999).

Model 7: Method to Measure Colonic Propulsive Motility in ExperimentalRodents.

This is used to demonstrate and characterize the pharmacological effectsof compounds on colonic propulsive motility in experimental rodents asdescribed (Martinez et al. J. Pharmacol. Experiment. Ther. 301: 611-617(2002)). The test is based on the reflex expulsion of a glass bead fromthe distal colon, which is indicative of drug effects on the reflex arc.This test is useful in evaluating whether diarrhea is a side effect.Mice or rats are fasted for one hour prior to administrations of thetest compound, such Zven polypeptides, such as Zven1, Zven2, as well asagonists, fragments, variants and/or chimeras thereof, or vehicle by theappropriate route (i.p., s.c., i.m., p.o., i.v.), followed 30 minutes(or other appropriate time) later by the insertion of a glass bead intothe distal colon. Rodents are marked for identification and placed inlarge glass beakers (or other) for observation. The time required forexpulsion of the bead is noted for each rodent.

Model 8: Model to Measure Gastrointestinal Motor Activity in Dogs.

Dogs are anesthetized and the abdominal cavity opened. Extraluminalforce transducers (sensor to measure contraction) are sutured onto five(5) sites, i.e., the gastric antrum, 3 cm proximal to the pyloric ring,the duodenum, 5 cm distal to the pyloric ring, the jejunum, 70 cm distalto the pyloric ring, the ileum, 5 cm proximal to the ileum-colonjunction, and the colon, 5 cm distal to the ileum-colon junction. Thelead wires of these force transducers are taken out of the abdominalcavity and then brought out through a skin incision made between thescapulae, at which a connector is connected. After the operation, ajacket protector is placed on the dog to protect the connector.Measurement of the gastrointestinal motor activity is started two weeksafter the operation. For ad libitum measurement, a telemeter(electrowave data transmitter) is connected with the connector todetermine the contractive motility at each site of the gastrointestinaltract. The data is stored in a computer via a telemeter for analysis. Atest compound, such as Zven polypeptides, such as Zven1, Zven2, as wellas agonists, fragments, variants and/or chimeras thereof, isadministered via the appropriate route (p.o., i.v., i.p., s.c., i.m.) atthe appropriate time point to assess its ability to affectgastrointestinal motor activity. This can be performed in normal dogs ordogs in which gastroparesis/ileus has been induced. The above method isa modification of those in Yoshida. and Ito. J. Pharmacol. Experiment.Therap. 257, 781-787 (1991) and Furuta et al. Biol. Pharm. Bull.25:103-1071 (2002).

12. Additional Methods to Measure Gastric Function

Model of Pain Assessment Associated with Gut Distention (in Rats;Rabbits; Dogs)

Indication: Inflammatory Bowel Disease (IBD), Irritable Bowl Syndrome(IBS), gastroparesis, ileus, dyspepsia.

Animals are surgically prepared with electrodes implanted on theproximal colon and striated muscles, and catheters implanted in lateralventricles of the brain. Rectal distension is performed by inflation ofa balloon rectally inserted, and the pressure eliciting a characteristicvisceromotor response is measured. A test compound, such as Zvenpolypeptides, such as Zven1, Zven2, as well as agonists, fragments,variants and/or chimeras thereof, is administered via the appropriateroute (p.o., i.p., s.c., i.v., or i.m.) and at the appropriate time(i.e. 20 min, if i.p. or i.c.v.) prior to distention. Test compound isevaluated for its ability to affect colonic motility, abdominalcontractions, and visceral pain.

Model to Assess Emesis (in Ferrets).

Indication: emesis (primary or as a result of gastroparesis)

The anti-emetic activity of a test compound is tested by its ability toinhibit cisplatin- or syrup of ipecac-induced emesis in the ferret(since mice and rats can not vomit). In this model the onset of retchingand vomiting occurs approximately 1 h after the administration ofcisplatin (200 mg/m.sup.2 i.p.). At the first retch in response tocisplatin, the test compound, Zven polypeptides, such as Zven1, Zven2,as well as agonists, fragments, variants and/or chimeras thereof, isadministered (e.g. i.p., p.o., i.v., s.c., i.c.v.) and its effect onemesis determined by comparison with appropriate controls (e.g. water).If using ipecac to induce emesis, the test compound may be given atappropriate time points prior to the ipecac. Latency to the first retch,the first vomit and the number of retching and vomiting episodes arerecorded over 60 min. Data are expressed as the mean latency (in min) tofirst retch or vomit; the mean number of emetic episodes per ferretbased on animals that did not exhibit emesis as well as those that did,and the mean number of retches/vomits exhibited by animals that remainedresponsive to ipecac (“responders”). Ferrets that fail to exhibit emesisare omitted from the latter calculation.

[i.e. (.+−.) cis-3-(2-methoxybenzylamino)-2-phenyl piperidine exhibitedanti-emetic activity when administered at a dose of 3 mg/kg i.p.]

Models of (Interstitial Cells of Cajal) ICC Loss.

Loss of ICC results in serious gastrointestinal motor dysfunction, andis seen in many diseases associated with altered gastrointestinalfunction. Antibodies to Kit provide the opportunity to evaluate ICCnetworks in gastrointestinal muscles in motility disorders.

Indications: diabetic gastroparesis; IBD; pseudo-obstruction, chronicconstipation.

Inducible Example: BALB/c mouse pups are treated with a monoclonalantibody (ACK2) to Kit for 4 d postnatally This suppresses thedevelopment of c-kit, resulting in a severe disorder of gut motility. Atest compound, Zven polypeptides, such as Zven1, Zven2, as well asagonists, fragments, variants and/or chimeras thereof, is administeredto assess its affect on gut motility. Isolated segments of the intestinefrom the Kit-treated mice may also be tested for rhythmic contractionand relaxation in vitro, in response to the test compound.

Spontaneous Example: The spontaneously diabetic NOD/LtJ mouse (JacksonLabs) develop delayed gastric emptying, impaired electrical pacemaking,and reduced motor neurotransmission. A test compound, Zven polypeptides,such as Zven1, Zven2, as well as agonists, fragments, variants and/orchimeras thereof, administered to assess its affect on gastric emptying(i.e. via phenol red/methyl cellulose) and gut motility. Isolatedsegments of the intestine from these mice may also be tested forrhythmic contraction and relaxation in vitro, in response to the testcompound administration.

Zven polypeptides, such as Zven1, Zven2, as well as agonists, fragments,variants and/or chimeras thereof, can also be used to increasesensitization in mammals. For example, an ortholog of Zven1 and Zven2,Bv8, was used to stimulate the PK-R1 and PK-R2 receptors in ratsresulting in sensitization of peripheral nociceptors to thermal andmechanic stimuli. See Negri, L. et al., Brit. J. Pharm. 137: 1147-1154,2002. Thus, the Zven1 and Zven2 polypeptides of the present invention,including agonists, can be used to increase sensitization (pain, heat,or mechanical) when delivered locally or topically, systemically, orcentrally. Also, the polypeptides of the present invention can beadministered to enhance the sensitivity of brain cells in order toimprove the function out of the surviving neurons to neurotransmittersand therefore might be effective in Parkinson's or Alzheimers disease.Zven1 polypeptides, and other Zven1 agonists, can also be used toalleviate pain, such as visceral pain or severe headache (e.g.,migraine).

Similarly, where a patient has an increased sensitization to pain,antagonists to Zven1 and Zven2 can be used to decrease the sensation ofpain in a patient with neuropathy. For example a patients with diabeticneuropathy have chronic, enhanced pain, the antagonist to zven1 may beuseful to limit, prevent or decrease the pain.

As shown in Example 5, Zven1 and Zven2 can stimulate angiogenesis.Accordingly, Zven1, Zven2, Zven1 agonists, and Zven2 agonists can beused to induce growth of new blood vessels. These molecules can beadministered to a mammalian subject alone or in combination with otherangiogenic factors, such as vascular endothelial growth factor.

Furthermore, prolonged gastrointestinal stasis often complicates thecourse of patients with sepsis (Hemann G, et al., Am. J. Phys. Regul.Integr. Compr. Physiol. 276:R59-R68, 1999). Activation of a systemicimmune response by injury, infection, radiation, or chemotherapy, isoften accompanied by gastric stasis which is perceived as nausea, lossof appetite and vomiting (Emch G., et al., Am. J. Physiol. Gastrointest.Liver Physiol. 279: G5582-G586, 2000). Thus, zven1 and it agonists maybe useful in treating sepsis related to gastrointestinal stasis orileus.

Additionally, such as Zven1, Zven2, as well as agonists, fragments,variants and/or chimeras thereof, may be useful in treating patientswith nausea and vomiting, especially where the nausea and vomiting arerelated to, or a result of ileus or other gastrointestinal motilitydisorders. These include when the vomiting is related to treatment forcancer, such as a prophylaxis, or post-administered, for chemotherapy.

Using telemetry in conscious male Sprague-Dawley rats, there were nosignificant changes in blood pressure or heart rate in response to ani.v. dose of 200 μg/kg Zven1. Stool consistency from these rats did notappear to be different during the 24 h period following Zven1administration. Rats do not appear to be affected by these doses ofZven1. There were no reported outward affects when mice wereadministered 10,000 μg/kg Zven1 via an i.p. injection.

The Zven polypeptides of the present invention, such as Zven1, Zven2, aswell as agonists, fragments, variants and/or chimeras thereof, can alsobe used as a supplement to food. Zven2 polypeptides have been purifiedfrom bovine milk. See Masuda Y. et al., Bioc. and Biophys. Res. Comm.293:396-402, 2002. Additionally, increased gastrointestinalcontractility can be conducive to improved metabolism and weight gain.As a protein that can be administered orally, Zven1 or Zven2, or acombination of agonists, variants, and/or fragments, can be useful as asupplement or adjuvant to a feeding program wherein the mammaliansubject suffers from a lack of appetite and/or weight gain. Suchconditions are known, for example, as failure to thrive, cachexia, andwasting syndromes. The polypeptides of the present invention may also beuseful adapting an infant mammal to digesting more conventional types offood.

Generally, the dosage of administered polypeptide, protein or peptidewill vary depending upon such factors as the patient's age, weight,height, sex, general medical condition and previous medical history.Typically, it is desirable to provide the recipient with a dosage of amolecule having Zven activity, which is in the range of from about 1pg/kg to 10 mg/kg (amount of agent/body weight of patient), although alower or higher dosage also may be administered as circumstancesdictate.

Zven1 polypeptides that were heated to 56 degrees C. for 30 minutesmaintained some activity, when measured by a reporter assay. See Example13. Thus, the polypeptides of the present invention may be effectivelydelivered orally.

Administration of a molecule having Zven activity to a subject can beintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, inhalation, as a suppository, or by direct intralesionalinjection. When administering therapeutic proteins by injection, theadministration may be by continuous infusion or by single or multipleboluses. Alternatively, Zven polypeptides, such as Zven1, Zven2, as wellas agonists, fragments, variants and/or chimeras thereof, can beadministered as a controlled release formulation.

Additional routes of administration include oral, dermal,mucosal-membrane, pulmonary, and transcutaneous. Oral delivery issuitable for polyester microspheres, zein microspheres, proteinoidmicrospheres, polycyanoacrylate microspheres, and lipid-based systems(see, for example, DiBase and Morrel, “Oral Delivery ofMicroencapsulated Proteins,” in Protein Delivery: Physical Systems,Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)). Thefeasibility of an intranasal delivery is exemplified by such a mode ofinsulin administration (see, for example, Hinchcliffe and Illum, Adv.Drug Deliv. Rev. 35:199 (1999)). Dry or liquid particles comprising suchas Zven1, Zven2, as well as agonists, fragments, variants and/orchimeras thereof, can be prepared and inhaled with the aid of dry-powderdispersers, liquid aerosol generators, or nebulizers (e.g., Pettit andGombotz, TIBTECH 16:343 (1998); Patton et al., Adv. Drug Deliv. Rev.35:235 (1999)). This approach is illustrated by the AERX diabetesmanagement system, which is a hand-held electronic inhaler that deliversaerosolized insulin into the lungs. Studies have shown that proteins aslarge as 48,000 kDa have been delivered across skin at therapeuticconcentrations with the aid of low-frequency ultrasound, whichillustrates the feasibility of trascutaneous administration (Mitragotriet al., Science 269:850 (1995)). Transdermal delivery usingelectroporation provides another means to administer such as Zven1,Zven2, as well as agonists, fragments, variants and/or chimeras thereof,(Potts et al., Pharm. Biotechnol. 10:213 (1997)).

Zven proteins can also be applied topically as, for example, liposomalpreparations, gels, salves, as a component of a glue, prosthesis, orbandage, and the like.

Since chemokines can promote and accelerate tissue repair, such asZven1, Zven2, as well as agonists, fragments, variants and/or chimerasthereof, can have a beneficial role in resolving disease. For example,topical administration is useful for wound healing applications,including the prevention of excess scaring and granulation tissue,prevention of keyloids, and prevention of adhesions following surgery.

A pharmaceutical composition comprising molecules having Zven1 or Zven2activity can be furnished in liquid form, in an aerosol, or in solidform. Proteins having Zven1 or Zven2 activity can be administered as aconjugate with a pharmaceutically acceptable water-soluble polymermoiety. As an illustration, a Zven1-polyethylene glycol conjugate isuseful to increase the circulating half-life of the interferon, and toreduce the immunogenicity of the polypeptide. Liquid forms, includingliposome-encapsulated formulations, are illustrated by injectablesolutions and oral suspensions. Exemplary solid forms include capsules,tablets, and controlled-release forms, such as a miniosmotic pump or animplant. Other dosage forms can be devised by those skilled in the art,as shown, for example, by Ansel and Popovich, Pharmaceutical DosageForms and Drug Delivery Systems, 5^(th) Edition (Lea & Febiger 1990),Gennaro (ed.), Remington's Pharmaceutical Sciences, 19^(th) Edition(Mack Publishing Company 1995), and by Ranade and Hollinger, DrugDelivery Systems (CRC Press 1996).

A pharmaceutical composition comprising a protein, polypeptide, orpeptide having Zven1 or Zven2 activity can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe therapeutic proteins are combined in a mixture with apharmaceutically acceptable carrier. A composition is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well-known to those in the art. See, for example, Gennaro(ed.), Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

For purposes of therapy, molecules having Zven1 or Zven2 activity and apharmaceutically acceptable carrier are administered to a patient in atherapeutically effective amount. A combination of a protein,polypeptide, or peptide having Zven activity and a pharmaceuticallyacceptable carrier is said to be administered in a “therapeuticallyeffective amount” if the amount administered is physiologicallysignificant. An agent is physiologically significant if its presenceresults in a detectable change in the physiology of a recipient patient.

For example, the present invention includes methods of increasing ordecreasing gastrointestinal contractility, gastric emptyint, and/orintestinal transt, comprising the step of administering a compositioncomprising a Zven2polypeptide, such as Zven1, Zven2, as well asagonists, fragments, variants and/or chimeras thereof, to the patient.In an in vivo approach, the composition is a pharmaceutical composition,administered in a therapeutically effective amount to a mammaliansubject.

A pharmaceutical composition comprising molecules having Zven activitycan be furnished in liquid form, or in solid form. Liquid forms,including liposome-encapsulated formulations, are illustrated byinjectable solutions and oral suspensions. Exemplary solid forms includecapsules, tablets, and controlled-release forms, such as a miniosmoticpump or an implant. Other dosage forms can be devised by those skilledin the art, as shown, for example, by Ansel and Popovich, PharmaceuticalDosage Forms and Drug Delivery Systems, 5^(th) Edition (Lea & Febiger1990), Gennaro (ed.), Remington's Pharmaceutical Sciences, 19^(th)Edition (Mack Publishing Company 1995), and by Ranade and Hollinger,Drug Delivery Systems (CRC Press 1996).

Zven1 or Zven2 pharmaceutical compositions may be supplied as a kitcomprising a container that comprises Zven1 or Zven2, a Zven1 or Zven2agonist, or a Zven1 or Zven2 antagonist (e.g., an anti-Zven1 or Zven2antibody or antibody fragment). For example, Zven1 or Zven2 can beprovided in the form of an injectable solution for single or multipledoses, or as a sterile powder that will be reconstituted beforeinjection. Alternatively, such a kit can include a dry-powder disperser,liquid aerosol generator, or nebulizer for administration of atherapeutic polypeptide. Such a kit may further comprise writteninformation on indications and usage of the pharmaceutical composition.Moreover, such information may include a statement that the Zven1 orZven2 composition is contraindicated in patients with knownhypersensitivity to Zven1 or Zven2.

13. Therapeutic Uses of Zven Nucleotide Sequences

The present invention includes the use of Zven nucleotide sequences toprovide Zven amino acid sequences to a subject in need of proteins,polypeptides, or peptides having Zven activity, as discussed in theprevious section. In addition, a therapeutic expression vector can beprovided that inhibits Zven gene expression, such as an anti-sensemolecule, a ribozyme, or an external guide sequence molecule.

There are numerous approaches to introduce a Zven gene to a subject,including the use of recombinant host cells that express Zven, deliveryof naked nucleic acid encoding Zven, use of a cationic lipid carrierwith a nucleic acid molecule that encodes Zven, and the use of virusesthat express Zven, such as recombinant retroviruses, recombinantadeno-associated viruses, recombinant adenoviruses, and recombinantHerpes simplex viruses [HSV] (see, for example, Mulligan, Science260:926 (1993), Rosenberg et al., Science 242:1575 (1988), LaSalle etal., Science 259:988 (1993), Wolff et al., Science 247:1465 (1990),Breakfield and Deluca, The New Biologist 3:203 (1991)). In an ex vivoapproach, for example, cells are isolated from a subject, transfectedwith a vector that expresses a Zven gene, and then transplanted into thesubject.

In order to effect expression of a Zven gene, an expression vector isconstructed in which a nucleotide sequence encoding a Zven gene isoperably linked to a core promoter, and optionally a regulatory element,to control gene transcription. The general requirements of an expressionvector are described above.

Alternatively, a Zven gene can be delivered using recombinant viralvectors, including for example, adenoviral vectors (e.g., Kass-Eisler etal., Proc. Nat'l Acad. Sci. USA 90:11498 (1993), Kolls et al., Proc.Nat'l Acad. Sci. USA 91:215 (1994), Li et al., Hum. Gene Ther. 4:403(1993), Vincent et al., Nat. Genet. 5:130 (1993), and Zabner et al.,Cell 75:207 (1993)), adenovirus-associated viral vectors (Flotte et al.,Proc. Nat'l Acad. Sci. USA 90:10613 (1993)), alphaviruses such asSemliki Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66:857(1992), Raju and Huang, J. Vir. 65:2501 (1991), and Xiong et al.,Science 243:1188 (1989)), herpes viral vectors (e.g., U.S. Pat. Nos.4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus vectors(Koering et al., Hum. Gene Therap. 5:457 (1994)), pox virus vectors(Ozaki et al., Biochem. Biophys. Res. Comm. 193:653 (1993), Panicali andPaoletti, Proc. Nat'l Acad. Sci. USA 79:4927 (1982)), pox viruses, suchas canary pox virus or vaccinia virus (Fisher-Hoch et al., Proc. Nat'lAcad. Sci. USA 86:317 (1989), and Flexner et al., Ann. N.Y. Acad. Sci.569:86 (1989)), and retroviruses (e.g., Baba et al., J. Neurosurg 79:729(1993), Ram et al., Cancer Res. 53:83 (1993), Takamiya et al., J.Neurosci. Res 33:493 (1992), Vile and Hart, Cancer Res. 53:962 (1993),Vile and Hart, Cancer Res. 53:3860 (1993), and Anderson et al., U.S.Pat. No. 5,399,346). Within various embodiments, either the viral vectoritself, or a viral particle which contains the viral vector may beutilized in the methods and compositions described below.

As an illustration of one system, adenovirus, a double-stranded DNAvirus, is a well-characterized gene transfer vector for delivery of aheterologous nucleic acid molecule (for a review, see Becker et al.,Meth. Cell Biol. 43:161 (1994); Douglas and Curiel, Science & Medicine4:44 (1997)). The adenovirus system offers several advantages including:(i) the ability to accommodate relatively large DNA inserts, (ii) theability to be grown to high-titer, (iii) the ability to infect a broadrange of mammalian cell types, and (iv) the ability to be used with manydifferent promoters including ubiquitous, tissue specific, andregulatable promoters. In addition, adenoviruses can be administered byintravenous injection, because the viruses are stable in thebloodstream.

Using adenovirus vectors where portions of the adenovirus genome aredeleted, inserts are incorporated into the viral DNA by direct ligationor by homologous recombination with a co-transfected plasmid. In anexemplary system, the essential E1 gene is deleted from the viralvector, and the virus will not replicate unless the E1 gene is providedby the host cell. When intravenously administered to intact animals,adenovirus primarily targets the liver. Although an adenoviral deliverysystem with an E1 gene deletion cannot replicate in the host cells, thehost's tissue will express and process an encoded heterologous protein.Host cells will also secrete the heterologous protein if thecorresponding gene includes a secretory signal sequence. Secretedproteins will enter the circulation from tissue that expresses theheterologous gene (e.g., the highly vascularized liver).

Moreover, adenoviral vectors containing various deletions of viral genescan be used to reduce or eliminate immune responses to the vector. Suchadenoviruses are E1-deleted, and in addition, contain deletions of E2Aor E4 (Lusky et al., J. Virol. 72:2022 (1998); Raper et al., Human GeneTherapy 9:671 (1998)). The deletion of E2b has also been reported toreduce immune responses (Amalfitano et al., J. Virol. 72:926 (1998)). Bydeleting the entire adenovirus genome, very large inserts ofheterologous DNA can be accommodated. Generation of so called “gutless”adenoviruses, where all viral genes are deleted, are particularlyadvantageous for insertion of large inserts of heterologous DNA (for areview, see Yeh. and Perricaudet, FASEB J. 11:615 (1997)).

High titer stocks of recombinant viruses capable of expressing atherapeutic gene can be obtained from infected mammalian cells usingstandard methods. For example, recombinant HSV can be prepared in Verocells, as described by Brandt et al., J. Gen. Virol. 72:2043 (1991),Herold et al., J. Gen. Virol. 75:1211 (1994), Visalli and Brandt,Virology 185:419 (1991), Grau et al., Invest. Ophthalmol. Vis. Sci.30:2474 (1989), Brandt et al., J. Virol. Meth. 36:209 (1992), and byBrown and MacLean (eds.), HSV Virus Protocols (Humana Press 1997).

Alternatively, an expression vector comprising a Zven gene can beintroduced into a subject's cells by lipofection in vivo usingliposomes. Synthetic cationic lipids can be used to prepare liposomesfor in vivo transfection of a gene encoding a marker (Felgner et al.,Proc. Nat'l Acad. Sci. USA 84:7413 (1987); Mackey et al., Proc. Nat'lAcad. Sci. USA 85:8027 (1988)). The use of lipofection to introduceexogenous genes into specific organs in vivo has certain practicaladvantages. Liposomes can be used to direct transfection to particularcell types, which is particularly advantageous in a tissue with cellularheterogeneity, such as the pancreas, liver, kidney, and brain. Lipidsmay be chemically coupled to other molecules for the purpose oftargeting. Targeted peptides (e.g., hormones or neurotransmitters),proteins such as antibodies, or non-peptide molecules can be coupled toliposomes chemically.

Electroporation is another alternative mode of administration of Zvennucleic acid molecules. For example, Aihara and Miyazaki, NatureBiotechnology 16:867 (1998), have demonstrated the use of in vivoelectroporation for gene transfer into muscle.

In an alternative approach to gene therapy, a therapeutic gene mayencode a Zven anti-sense RNA that inhibits the expression of Zven.Suitable sequences for Zven anti-sense molecules can be derived from thenucleotide sequences of Zven disclosed herein.

Alternatively, an expression vector can be constructed in which aregulatory element is operably linked to a nucleotide sequence thatencodes a ribozyme. Ribozymes can be designed to express endonucleaseactivity that is directed to a certain target sequence in a mRNAmolecule (see, for example, Draper and Macejak, U.S. Pat. No. 5,496,698,McSwiggen, U.S. Pat. No. 5,525,468, Chowrira and McSwiggen, U.S. Pat.No. 5,631,359, and Robertson and Goldberg, U.S. Pat. No. 5,225,337). Inthe context of the present invention, ribozymes include nucleotidesequences that bind with Zven mRNA.

In another approach, expression vectors can be constructed in which aregulatory element directs the production of RNA transcripts capable ofpromoting RNase P-mediated cleavage of mRNA molecules that encode a Zvengene. According to this approach, an external guide sequence can beconstructed for directing the endogenous ribozyme, RNase P, to aparticular species of intracellular mRNA, which is subsequently cleavedby the cellular ribozyme (see, for example, Altman et al., U.S. Pat. No.5,168,053, Yuan et al., Science 263:1269 (1994), Pace et al.,international publication No. WO 96/18733, George et al., internationalpublication No. WO 96/21731, and Werner et al., internationalpublication No. WO 97/33991). Preferably, the external guide sequencecomprises a ten to fifteen nucleotide sequence complementary to ZvenmRNA, and a 3′-NCCA nucleotide sequence, wherein N is preferably apurine. The external guide sequence transcripts bind to the targetedmRNA species by the formation of base pairs between the mRNA and thecomplementary external guide sequences, thus promoting cleavage of mRNAby RNase P at the nucleotide located at the 5′-side of the base-pairedregion.

In general, the dosage of a composition comprising a therapeutic vectorhaving a Zven nucleotide acid sequence, such as a recombinant virus,will vary depending upon such factors as the subject's age, weight,height, sex, general medical condition and previous medical history.Suitable routes of administration of therapeutic vectors includeintravenous injection, intraarterial injection, intraperitonealinjection, intramuscular injection, intratumoral injection, andinjection into a cavity that contains a tumor.

A composition comprising viral vectors, non-viral vectors, or acombination of viral and non-viral vectors of the present invention canbe formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby vectors or viruses are combined in amixture with a pharmaceutically acceptable carrier. As noted above, acomposition, such as phosphate-buffered saline is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient subject. Other suitable carriers are well-knownto those in the art (see, for example, Remington's PharmaceuticalSciences, 19th Ed. (Mack Publishing Co. 1995), and Gilman's thePharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co.1985)).

For purposes of therapy, a therapeutic gene expression vector, or arecombinant virus comprising such a vector, and a pharmaceuticallyacceptable carrier are administered to a subject in a therapeuticallyeffective amount. A combination of an expression vector (or virus) and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient subject.

When the subject treated with a therapeutic gene expression vector or arecombinant virus is a human, then the therapy is preferably somaticcell gene therapy. That is, the preferred treatment of a human with atherapeutic gene expression vector or a recombinant virus does notentail introducing into cells a nucleic acid molecule that can form partof a human germ line and be passed onto successive generations (i.e.,human germ line gene therapy).

14. Detection of Zven1 Gene Expression with Nucleic Acid Probes

Nucleic acid molecules can be used to detect the expression of a Zven1or Zven2 gene in a biological sample. Such probe molecules includedouble-stranded nucleic acid molecules comprising the nucleotidesequence of SEQ ID NO:1, or a fragment thereof, as well assingle-stranded nucleic acid molecules having the complement of thenucleotide sequence of SEQ ID NO:1, or a fragment thereof. Probemolecules may be DNA, RNA, oligonucleotides, and the like.

Illustrative probes comprise a portion of the nucleotide sequence ofnucleotides 66 to 161 of SEQ ID NO:1, the nucleotide sequence ofnucleotides 288 to 389 of SEQ ID NO:1, or the complement of suchnucleotide sequences. An additional example of a suitable probe is aprobe consisting of nucleotides 354 to 382 of SEQ ID NO:1, or a portionthereof. As used herein, the term “portion” refers to at least eightnucleotides to at least 20 or more nucleotides.

For example, nucleic acid molecules comprising a portion of thenucleotide sequence of SEQ ID NO:1 can be used to detect activatedneutrophils. Such molecules can also be used to identity therapeutic orprophylactic agents that modulate the response of a neutrophil to apathogen.

In a basic detection assay, a single-stranded probe molecule isincubated with RNA, isolated from a biological sample, under conditionsof temperature and ionic strength that promote base pairing between theprobe and target Zven1 RNA species. After separating unbound probe fromhybridized molecules, the amount of hybrids is detected.

Well-established hybridization methods of RNA detection include northernanalysis and dot/slot blot hybridization (see, for example, Ausubel(1995) at pages 4-1 to 4-27, and Wu et al. (eds.), “Analysis of GeneExpression at the RNA Level,” in Methods in Gene Biotechnology, pages225-239 (CRC Press, Inc. 1997)). Nucleic acid probes can be detectablylabeled with radioisotopes such as ³²P or ³⁵ S. Alternatively, Zven RNAcan be detected with a nonradioactive hybridization method (see, forexample, Isaac (ed.), Protocols for Nucleic Acid Analysis byNonradioactive Probes (Humana Press, Inc. 1993)). Typically,nonradioactive detection is achieved by enzymatic conversion ofchromogenic or chemiluminescent substrates. Illustrative nonradioactivemoieties include biotin, fluorescein, and digoxigenin.

Zven1 oligonucleotide probes are also useful for in vivo diagnosis. Asan illustration, ¹⁸F-labeled oligonucleotides can be administered to asubject and visualized by positron emission tomography (Tavitian et al.,Nature Medicine 4:467 (1998)).

Numerous diagnostic procedures take advantage of the polymerase chainreaction (PCR) to increase sensitivity of detection methods. Standardtechniques for performing PCR are well-known (see, generally, Mathew(ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),White (ed.), PCR Protocols: Current Methods and Applications (HumanaPress, Inc. 1993), Cotter (ed.), Molecular Diagnosis of Cancer (HumanaPress, Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols(Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR(Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis (HumanaPress, Inc. 1998)).

One variation of PCR for diagnostic assays is reverse transcriptase-PCR(RT-PCR). In the RT-PCR technique, RNA is isolated from a biologicalsample, reverse transcribed to cDNA, and the cDNA is incubated withZven1 primers (see, for example, Wu et al. (eds.), “Rapid Isolation ofSpecific cDNAs or Genes by PCR,” in Methods in Gene Biotechnology, pages15-28 (CRC Press, Inc. 1997)). PCR is then performed and the productsare analyzed using standard techniques.

As an illustration, RNA is isolated from biological sample using, forexample, the guanidinium-thiocyanate cell lysis procedure describedabove. Alternatively, a solid-phase technique can be used to isolatemRNA from a cell lysate. A reverse transcription reaction can be primedwith the isolated RNA using random oligonucleotides, short homopolymersof dT, or Zven1 anti-sense oligomers. Oligo-dT primers offer theadvantage that various mRNA nucleotide sequences are amplified that canprovide control target sequences. Zven1 sequences are amplified by thepolymerase chain reaction using two flanking oligonucleotide primersthat are typically 20 bases in length.

PCR amplification products can be detected using a variety ofapproaches. For example, PCR products can be fractionated by gelelectrophoresis, and visualized by ethidium bromide staining.Alternatively, fractionated PCR products can be transferred to amembrane, hybridized with a detectably-labeled Zven1 probe, and examinedby autoradiography. Additional alternative approaches include the use ofdigoxigenin-labeled deoxyribonucleic acid triphosphates to providechemiluminescence detection, and the C-TRAK colorimetric assay.

Another approach for detection of Zven1 expression is cycling probetechnology (CPT), in which a single-stranded DNA target binds with anexcess of DNA-RNA-DNA chimeric probe to form a complex, the RNA portionis cleaved with RNAase H, and the presence of cleaved chimeric probe isdetected (see, for example, Beggs et al., J. Clin. Microbiol. 34:2985(1996), Bekkaoui et al., Biotechniques 20:240 (1996)). Alternativemethods for detection of Zven1 sequences can utilize approaches such asnucleic acid sequence-based amplification (NASBA), cooperativeamplification of templates by cross-hybridization (CATCH), and theligase chain reaction (LCR) (see, for example, Marshall et al., U.S.Pat. No. 5,686,272 (1997), Dyer et al., J. Virol. Methods 60:161 (1996),Ehricht et al., Eur. J. Biochem. 243:358 (1997), and Chadwick et al., J.Virol. Methods 70:59 (1998)). Other standard methods are known to thoseof skill in the art.

Zven1 probes and primers can also be used to detect and to localizeZven1 gene expression in tissue samples. Methods for such in situhybridization are well-known to those of skill in the art (see, forexample, Choo (ed.), In Situ Hybridization Protocols (Humana Press, Inc.1994), Wu et al. (eds.), “Analysis of Cellular DNA or Abundance of mRNAby Radioactive In Situ Hybridization (RISH),” in Methods in GeneBiotechnology, pages 259-278 (CRC Press, Inc. 1997), and Wu et al.(eds.), “Localization of DNA or Abundance of mRNA by Fluorescence InSitu Hybridization (RISH),” in Methods in Gene Biotechnology, pages279-289 (CRC Press, Inc. 1997)). Various additional diagnosticapproaches are well-known to those of skill in the art (see, forexample, Mathew (ed.), Protocols in Human Molecular Genetics (HumanaPress, Inc. 1991), Coleman and Tsongalis, Molecular Diagnostics (HumanaPress, Inc. 1996), and Elles, Molecular Diagnosis of Genetic Diseases(Humana Press, Inc., 1996)).

15. Detection of Zven1 Protein with Anti-Zven1 Antibodies

The present invention contemplates the use of anti-Zven1 antibodies toscreen biological samples in vitro for the presence of Zven1, andparticularly for the upregulation of Zven1. In one type of in vitroassay, anti-Zven1 antibodies are used in liquid phase. For example, thepresence of Zven1 in a biological sample can be tested by mixing thebiological sample with a trace amount of labeled Zven1 and an anti-Zven1antibody under conditions that promote binding between Zven1 and itsantibody. Complexes of Zven1 and anti-Zven1 in the sample can beseparated from the reaction mixture by contacting the complex with animmobilized protein which binds with the antibody, such as an Fcantibody or Staphylococcus protein A. The concentration of Zven1 in thebiological sample will be inversely proportional to the amount oflabeled Zven1 bound to the antibody and directly related to the amountof free-labeled Zven1. Anti-Zven2 antibodies can be used in the same ora similar fashion.

Alternatively, in vitro assays can be performed in which anti-Zven1antibody is bound to a solid-phase carrier. For example, antibody can beattached to a polymer, such as aminodextran, in order to link theantibody to an insoluble support such as a polymer-coated bead, a plateor a tube. Other suitable in vitro assays will be readily apparent tothose of skill in the art.

In another approach, anti-Zven1 antibodies can be used to detect Zven1in tissue sections prepared from a biopsy specimen. Such immunochemicaldetection can be used to determine the relative abundance of Zven1 andto determine the distribution of Zven1 in the examined tissue. Generalimmunochemistry techniques are well established (see, for example,Ponder, “Cell Marking Techniques and Their Application,” in MammalianDevelopment: A Practical Approach, Monk (ed.), pages 115-38 (IRL Press1987), Coligan at pages 5.8.1-5.8.8, Ausubel (1995) at pages 14.6.1 to14.6.13 (Wiley Interscience 1990), and Manson (ed.), Methods InMolecular Biology, Vol. 10: Immunochemical Protocols (The Humana Press,Inc. 1992)).

Immunochemical detection can be performed by contacting a biologicalsample with an anti-Zven1 antibody, and then contacting the biologicalsample with a detectably labeled molecule that binds to the antibody.For example, the detectably labeled molecule can comprise an antibodymoiety that binds to anti-Zven1 antibody. Alternatively, the anti-Zven1antibody can be conjugated with avidin/streptavidin (or biotin) and thedetectably labeled molecule can comprise biotin (oravidin/streptavidin). Numerous variations of this basic technique arewell-known to those of skill in the art.

Alternatively, an anti-Zven1 antibody can be conjugated with adetectable label to form an anti-Zven1 immunoconjugate. Suitabledetectable labels include, for example, a radioisotope, a fluorescentlabel, a chemiluminescent label, an enzyme label, a bioluminescent labelor colloidal gold. Methods of making and detecting suchdetectably-labeled immunoconjugates are well-known to those of ordinaryskill in the art, and are described in more detail below.

The detectable label can be a radioisotope that is detected byautoradiography. Isotopes that are particularly useful for the purposeof the present invention are ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C.

Anti-Zven1 immunoconjugates can also be labeled with a fluorescentcompound. The presence of a fluorescently-labeled antibody is determinedby exposing the immunoconjugate to light of the proper wavelength anddetecting the resultant fluorescence. Fluorescent labeling compoundsinclude fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

Alternatively, anti-Zven1 immunoconjugates can be detectably labeled bycoupling an antibody component to a chemiluminescent compound. Thepresence of the chemiluminescent-tagged immunoconjugate is determined bydetecting the presence of luminescence that arises during the course ofa chemical reaction. Examples of chemiluminescent labeling compoundsinclude luminol, isoluminol, an aromatic acridinium ester, an imidazole,an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label anti-Zven1immunoconjugates of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Bioluminescent compounds that are useful forlabeling include luciferin, luciferase and aequorin.

Alternatively, anti-Zven1 immunoconjugates can be detectably labeled bylinking an anti-Zven1 antibody component to an enzyme. When theanti-Zven1-enzyme conjugate is incubated in the presence of theappropriate substrate, the enzyme moiety reacts with the substrate toproduce a chemical moiety, which can be detected, for example, byspectrophotometric, fluorometric or visual means. Examples of enzymesthat can be used to detectably label polyspecific immunoconjugatesinclude β-galactosidase, glucose oxidase, peroxidase and alkalinephosphatase.

Those of skill in the art will know of other suitable labels, which canbe employed in accordance with the present invention. The binding ofmarker moieties to anti-Zven1 antibodies can be accomplished usingstandard techniques known to the art. Typical methodology in this regardis described by Kennedy et al., Clin. Chim. Acta 70:1 (1976), Schurs etal., Clin. Chim. Acta 81:1 (1977), Shih et al., Int'l Cancer 46:1101(1990), Stein et al., Cancer Res. 50:1330 (1990), and Coligan, supra.

Moreover, the convenience and versatility of immunochemical detectioncan be enhanced by using anti-Zven1 antibodies that have been conjugatedwith avidin, streptavidin, and biotin (see, for example, Wilchek et al.(eds.), “Avidin-Biotin Technology,” Methods In Enzymology, Vol. 184(Academic Press 1990), and Bayer et al., “Immunochemical Applications ofAvidin-Biotin Technology,” in Methods In Molecular Biology, Vol. 10,Manson (ed.), pages 149-162 (The Humana Press, Inc. 1992).

Methods for performing immunoassays are well-established. See, forexample, Cook and Self, “Monoclonal Antibodies in DiagnosticImmunoassays,” in Monoclonal Antibodies: Production, Engineering, andClinical Application, Ritter and Ladyman (eds.), pages 180-208,(Cambridge University Press, 1995), Perry, “The Role of MonoclonalAntibodies in the Advancement of Immunoassay Technology,” in MonoclonalAntibodies: Principles and Applications, Birch and Lennox (eds.), pages107-120 (Wiley-Liss, Inc. 1995), and Diamandis, Immunoassay (AcademicPress, Inc. 1996).

In a related approach, biotin- or FITC-labeled Zven1 can be used toidentify cells that bind Zven1. Such can binding can be detected, forexample, using flow cytometry.

The present invention also contemplates kits for performing animmunological diagnostic assay for Zven1 gene expression. Such kitscomprise at least one container comprising an anti-Zven1 antibody, orantibody fragment. A kit may also comprise a second container comprisingone or more reagents capable of indicating the presence of Zven1antibody or antibody fragments. Examples of such indicator reagentsinclude detectable labels such as a radioactive label, a fluorescentlabel, a chemiluminescent label, an enzyme label, a bioluminescentlabel, colloidal gold, and the like. A kit may also comprise a means forconveying to the user that Zven1 antibodies or antibody fragments areused to detect Zven1 protein. For example, written instructions maystate that the enclosed antibody or antibody fragment can be used todetect Zven1. The written material can be applied directly to acontainer, or the written material can be provided in the form of apackaging insert.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention. The examples describe studies using Zven1 protein produced inbaculovirus with a C-terminal Glu-Glu tag, following the methodsgenerally described above. Zven2 (“endocrine-gland-derived vascularendothelial growth factor”) protein was purchased from Peprotech, Inc.(Rocky Hill, N.J.).

16. EXAMPLES Example 1 Stimulation of Responses in Wky12-22 Cells

Wky12-22 cells were derived from the medial layer of the thoracic aortaof Wistar-Kyoto rat pups, as described by Lemire et al., AmericanJournal of Pathology 144:1068 (1994). These cells respond to both Zven1and Zven2 in a reporter luciferase assay following transfection withNFkB/Ap-1 reporter construct. A control cell line, Wky3M-22, derivedfrom the same tissue in adult rat did not signal. Activity was detectedat concentrations ranging from 1-100 ng/ml of Zven1 or Zven2(approximately 0.1 nM-10 nM). These data suggest that Wky12-22 cellscarry the Zven1 receptor, and that Zven1 and Zven2 activate theNfKb/Ap-1 transcription factor.

In one experiment, Wky12-22 cells were loaded with the fluorescent dyeFura. The emission peak of Fura shifts when bound to calcium.Intracellular calcium release is detected by monitoring the wavelengthshift. Zven1 induced intracellular calcium release at concentrations of1-1000 ng/ml. Zven2 induced a similar response.

Extracellular signal-regulated kinase/mitogen-activated protein kinase(ERK-Map kinase) activity was measured in Wky12-22 cells in response toZven1 treatment. Cells were incubated in Zven1 at concentrations rangingfrom 1 to 1,000 ng/ml for thirty minutes. Cells were fixed and stainedfor phosphorylated ERK-Map kinase using the Arrayscan, which measuresthe fluorescent intensities in the cytosol and the nucleus of thetreated cell. The difference in fluorescence of the nucleus and thecytosol were quantified and plotted. Zven1 induced ERK-Map kinaseactivity with an EC₅₀ of 0.50 nM (approximately 5 ng/ml).

The binding of Zven1 to Wky12-22 cells was assessed usingI¹²⁵-radiolabeled Zven1. Wky12-22 cells were seeded at low cell densityand cultured for three to four days until they reached about 70%confluency. The cells were placed on ice, the medium was removed, andthe monolayers were washed. The cells were incubated with increasingamounts of I¹²⁵-Zven1 in the absence (total binding) and presence(nonspecific binding) of a large excess of unlabeled Zven1. Aftervarious times at 4° C., the binding media were removed, the monolayerswere washed, and the cells were solubilized with a small volume of 1.0 NNaOH. Cell associated radioactivity was determined in a gamma counter.The specific binding of I¹²⁵-Zven1 was calculated as the differencebetween the total and nonspecific values. The measured radioacitivitywas normalized to cell number that was determined on a set of parallelcultures. Nonlinear regression using a two-site model was used to fitthe binding data for determination of Kd and Bmax. The high affinitysite exhibited a Kd of 1.5 nM and a Bmax of 350 fmol bound/10⁶ cellswhereas the low affinity site showed a Kd of 31 nM with a Bmax of 1025fmol bound/10⁶ cells.

The results of these studies show that a neonatal rat aortic cellexpresses the Zven1 receptor while equivalent adult rat cells do not.This suggests that Zven1 is involved with heart development andvasculogenesis. Zven1 signals through NFkB/Ap1 and induces chemokinerelease only in the neonatal cells, suggesting that it may trigger amitogenic response in fetal or neonatal heart. Zven1 may be a requiredfactor necessary for the induction of vasculogenesis/angiogenesis incardiac stem cells. Zven1 induces intracellular calcium release in theWky12-22 cell line, an effect consistent with chemokine activity.Consistent with its mitogenic activity, Zven1 activates a mitogenactivated protein kinase.

Example 2 Zven1 and Zven2 Stimulate Chemokine Release In Vitro

Confluent Wky12-22 or Wky3M22 cells were incubated with varyingconcentrations of Zven1 for twenty-four hours. Conditioned media werecollected and assayed for the chemokine CINC-1 using acommercially-available rat cytokine multiplex kit (Linco Research, Inc.;St. Charles, Mo.). CINC-1, thought to be equivalent to humangrowth-related oncogene-α (GRO-α), was detected at levels ranging from1.8-5 ng/ml in cells treated with 0.1 to 100 ng/ml of Zven1respectively. Zven2 induced an equivalent level of CINC-1 release fromWky12-22 cells. CINC-1 was not detected in either the control Wky3M-22cell line derived from adult rat aorta, or non-treated controls.

Example 3 Zven1 Induces a Chemotactic Response and Stimulates ChemokineRelease and Neutrophil Infiltration In Vivo

Four groups of ten mice (BALB57/BL6 females at eight weeks of age) wereeither not treated, or injected with vehicle buffer control, 0.1 μg ofZven1 or 1 μg of Zven1. Four hours later, peritoneal lavage fluid wascollected, concentrated, and the cell pellets were resuspended. Therelative cell populations were enumerated using the Cell Dyne, andcytospins were prepared for CBC/diff counts. The non-treated and buffercontrol animals had approximately 2% neutrophils in their lavage fluid,while the 0.1 μg treated animals had approximately 30% neutrophils,indicating an approximate 15-fold increase in neutrophils in theperitoneum of the Zven1-treated animals. The 1 μg Zven1-treated animalshad neutrophil levels consistent with the non-treated controls,suggesting a biphasic Zven1 response. In sum, Zven1 induced neutrophilinfiltration into the peritoneum following intraperitoneal injection.

Murine KC, the ortholog of GROα in mice, was measured in serum andlavage fluids obtained from the four groups of mice using an ELISA kit(R&D Systems Inc.; MN). The 0.1μg Zven1-treated (low dose) mice hadapproximately 45 picograms/ml KC in their peritoneal fluid, which wassignificantly higher than the non-treated controls, the vehiclecontrols, and the 1.0 μg Zven1-treated (high dose) mice.

Serum levels of KC in the 0.1 μg Zven1-treated mice were considerablyhigher than the non-treated, the 1.0 μg Zven1-treated, and thevehicle-treated mice. The 0.1 μg Zven1-treated mice had KC levels ofapproximately 185 picograms/ml, which is a six-fold increase.

TABLE 5 Murine KC in Zven1-treated mice following IP injectionConcentration of Murine KC (picogram/ml) Non-treated Vehicle 0.1 μg 1.0μg animals Control Zven1/animal Zven1/animal Lavage Fluid 10 21 45 8Serum 30 38 185 50

These results are consistent with the stimulation of chemokine releasein vitro shown in Example 2. Furthermore these results correlate withthe observed neutriphil infiltration in the peritoneum in the 0.1 μgZven1-treated (low dose) mice.

Example 4 Zven1 Effect on Gastric Emptying

Seven mice received an intraperitoneal injection of approximately 200 μgof Zven1 (10 μg/g body weight) or vehicle control followed by 7.5 mgphenol red. Gastric function was measured by monitoring phenol redtransport through the gut after twenty minutes. The general behavior ofZven1 treated animals was observed and was consistent with the behaviorof the control animals. In the Zven1-treated mice, gastric transit timewas reduced by approximately 50%.

These results show that, at high doses following intraperitonealinjection, Zven1 reduces gastric transit. Zven1 administration did notappear to have any immediate toxic effects. This reduction in transitmay be the result of a massive muscle contraction at such high doses.Zven1 may well increase motility in vivo at low doses, and inhibitmotility at high doses.

Example 5 Stimulation of Angiogenesis by Zven1 and Zven2

Thoracic aortas were removed from twelve-day, five-week, and three-monthold Wistar rats. The tissues were flushed with Hanks basic salt solutionto remove any blood cells and adventitial tissues were removed. Aorticrings were prepared and plated on Matrigel coated plates in serum freemodified MCDB media from Clonetics plus antibiotics,penicillin-streptomycin. Varying concentrations of Zven1 and Zven2 wereadded to culture dish approximately thirty minutes after plating.Proliferation was measured visually and individual rings werephotographed to record results. Both Zven1 and Zven2 induced aproliferative response at concentrations ranging from 1 to 100 ng/ml.This mitogenic effect was observed in aortas from the animals at allthree ages. Zven1 was also tested in the rat corneal model ofanigiogenesis where no effect was noted. The observed angiogenic effectin the aortic ring cultures may be due to the mitogenic effects of theGROα homologue.

Example 6 Stimulation of Contractility in Guinea Pig GastrointestinialOrgan Bath Assay

Male Hartley Guinea pigs at six weeks of age weighing approximately 0.5kg were euthanized by carbon monoxide. Intestinal tissue was harvestedas follows: 2-3 cm longitudinal sections of ileum 10 cm rostral of thececum, and 2-3 cm longitudinal sections of duodenum, jejunum, andproximal and distal colon.

Tissue was washed in Krebs Ringer's Bicarbonate buffer containing 118.2mM NaCl, 4.6 mM KCl, 1.2 mm MgS0₄, 24.8 mM NaHC0₃, 1.2 mM KH₂P0₄, 2.5 mMCaCl₂ and 10 mM glucose. Following a thorough wash, the tissue wasmounted longitudinally in a Radnoti organ bath perfusion system (SDRClinical Technology, Sydney Australia) containing oxygenated Krebsbuffer warmed and maintained at 37° C. A one gram pre-load was appliedand the tissue strips were allowed to incubate for approximately 30minutes. Baseline contractions were then obtained. Isometriccontractions were measured with a force displacement transducer andrecorded on a chart recorder using Po-ne-mah Physiology PlatformSoftware. The neurotransmitter 5 Hydroxytryptophane (5HT) (Sigma) at 130μm, and atropine at 5-10 mM were used as controls. Atropine blocks themuscarinic effect of acetylcholine.

Varying doses of Zven1 from 1-400 ng/ml were tested for activity onstrips of ileum. Muscle contractions were detected immediately afteradding zven1 protein and were recorded at concentrations as low as 1ng/ml or 100 picomolar. The EC 50 of this response was approximately 10ng/ml or 1 nM. Zven1 was tested for activity in the presence of 5HT, anda secondary contraction was observed. Zven1 was tested for activity inthe presence of 0.1 μM tetrodotoxin (TTX), the nerve action potentialantagonist and no reduction in the zven1 effect was observed. Zven1 wasalso tested for activity in the presence of 100 nM Verapamil, the L-typecalcium channel blocker. A significant reduction in the amplitude of thecontractile response was observed.

Results of the effect of zven1 on contractions in the ileum are shown inTable 6.

TABLE 6 Summary of Ileum Organ Bath Test Results Treatment Ileum 40ng/ml Zven1 +C 40 ng/ml +C Zven1 + 130 μM 5HT 40 ng/ml Zven1 + 5 mM +CAtropine 40 ng/ml Zven1 + 1 μM — Verapamil 40 ng/nL Zven1 + 0.1 μM +CTTX +C = Contraction Observed — = No zven1 effect observed

Results of the effect of zven1 on contractions in duodenum, jejunum,proximal colon, and distal colon were performed at a concentration of 40ng/ml did not produce contractions in duodenum, jejunum, or distalcolon. However, relaxation of the tissue of the proximal colon wasobserved when the same concentration of zven1 was added.

Example 7 Effect of Dose on Contractility in Guinea Pig Ileal Organ BathAssay

All intestinal sections from the guinea pig ileum were obtained andtested using the same protocol and reagents as described in Example 6.Longitudinal strips of guinea pig ileum were mounted in the organ bathand allowed to stabilize for approximately 20 minutes. Acetylcholine(ACH) at a concentration of 10 μg/ml was added to tissue to confirmcontractile activity. Two flush and fill cycles were run to wash ACHfrom the intestinal tissue. Baseline activity was confirmed forapproximately 25 minutes. Zven1 was added to the organ bath at a finalconcentration of 1.0 ng/ml and an approximate 0.5 gram of deflection wasrecorded. The 1.0 ng/ml zven1 dose was left on the tissue for 5 minutesto allow the tissue to return to baseline levels, and then a 10 ng/mldose was added. Another contractile response was noted that resulted ina 2.0 gram deflection. The 10 ng/ml dose was left on for another 5minutes before dosing the tissue with a 20 ng/ml dose of zven1. Anothercontractile response was observed, yielding an approximate 2.2 gramdeflection. Following a 5 minute incubation, the tissue was treated witha 40 ng/ml dose of zven1. The tissue contracted again, with anapproximate 2.0 gram deflection. The highest response was observed atthe 20 ng/mL zven1 dose.

Example 8 Effect of Zven1 on Gastric Emptying and Intestinal Transit

Eight-week old female C57B1/6 mice were fed a test meal consisting of amethylcellulose solution or a control, and both gastric emptying andintestinal transit was measured by determining the amount of phenol redrecovered in different sections of the intestine. The test meal consistsof a 1.5% aqueous methylcellulose solution containing a non-absorbabledye, 0.05% phenol red (50 mg/100 ml Sigma Chemical Company Catalogue #P4758). Medium viscosity carboxy methylcellulose from Sigma (Catalogue#C4888) with a final viscosity of 400-800 centipoises was used. Onegroup of animals was sacrificed immediately following administration oftest meal. These animals represent the standard group, 100% phenol redin stomach or Group VIII. The remaining animals were sacrificed 20minutes post administration of test meal. Following sacrifice, thestomach was removed and the small intestine was sectioned into proximal,mid and distal gut sections. The proximal gut consisted approximately ofduodenum, the mid gut consisted approximately of duodenum and jejunum,and the distal gut consisted approximately of ileum. All tissues weresolubilized in 10 mls of 0.1 N NaOH using a tissue homogenizer.Spectrophotometric analysis was used to determine the OD and hence thelevel of gastric emptying and gut transit.

Each treatment group consisted of 10 animals, except for the animalsbeing used as a standard group and the caerulein control group where then=5. The study was broken down into two days, such that one half of alltreatment groups are done on two consecutive days. The animals werefasted for 18 hrs in elevated cages, allowing access to water. Theaverage weight of the mice was 16 grams.

Baculovirus-expressed Zven1 protein with a C-terminal Glu-Glu tagformulated in 20 mM MES buffer, 20 mM NaCl, pH 6.5 was diluted into 0.9%NaCl+0.1% BSA using siliconized tubes. (Sigma sodium chloride solution0.9%, and Sigma BSA 30% sterile TC tested solution, Sigma Chemical Co,St Louis, Mo.). The protein concentration was adjusted so as to becontained in a 0.2 ml volume per mouse. Vehicle animals received anequivalent dose of zven1 formulation buffer based on the highest (775ng/g) treatment group.

Treatments were administered in a 0.2 ml volume via IP (intraperitoneal)injection two minutes prior to receiving 0.15 ml phenol red test meal asan oral gavage. Twenty minutes post administration of phenol red,animals were euthanized and stomach and intestinal segments removed. Theintestine was measured and divided into three equal segments: proximal,mid and distal gut. The amount of phenol red in each sample wasdetermined by spectrophotometric analysis and expressed as the percentof total phenol red in the stomach (Group VIII). These values were usedto determine the amount of gastric emptying and gut transit per tissuecollected. The CCK analogue caerulein at 40 ng/gram was used as apositive control and was administered five minutes prior to gavage, atwhich concentration it inhibits gastric emptying. Colormetric analysisof phenol red recovered from each gut segment and stomach was performedas follows. After euthanization, the stomach and intestinal segmentswere placed into 10 mls of 0.1 N NaOH and homogenized using a polytrontissue homogenizer. The homogenate was incubated for 1 hour at roomtemperature then pelleted by centrifugation on a table top centrifuge at150×g for 20 minutes at 4 degrees C. Proteins were precipitated from 5.0mls of the homogenate by the addition of 0.5 ml of 20% trichloraceticacid. Following centrifugation, 4 mls of supernatant was added to 4 mlsof 0.5 N NaOH. A 200 μl sample was read at 560 nm using MolecularDevices Spectra Max 190 spectrophotometer. The amount of gastricemptying was calculated using the following formula: percent gastricemptying=(1−amount phenol red recovered from test stomach/average amountof phenol red recovered from Group VII stomach)×100. The amount ofgastric transit was expressed as the percent of total phenol redrecovered.

Results are shown in Table 7, below. Since test meal was not detected inthe distal gut under any conditions, these data are not included. Asexpected, caerulein at 40 ng/ml inhibited gastric emptying (93.8% oftest meal in stomach after 20 minutes compared to 63.8% with vehicle).Consistent with inhibited gastric emptying, in the caerulein treatedgroup only 2.6% of meal was measured in the proximal gut and 1.2% in themid gut.

At the lowest zven1 concentration, 0.78 ug/kg body weight, a slightincrease in gastric emptying compared to vehicle was observed (56.3% ofmeal remaining versus 63.8% with vehicle). Consistent with an increasein gastric emptying, increased meal was detected in the proximal gut ofthe zven1 treated animals compared to vehicle control, 25.5% and 18.4%respectively. At the 7.8 ug/kg dose, zven1 treated animals had 20% lesstest meal in the stomach (p=0.001), 16.6% more meal in the proximal gut(p=0.004) and 3.5% more meal in the mid gut. The largest effect wasobserved with the 77.5 ug/kg animals where gastric emptying wasincreased approximately 2 fold (37.8% test meal in zven1 treated animalsand 63.8% in vehicle treated animals p=0.0002). Intestinal transit wasalso increased significantly as a greater than 2 fold increase in testmeal in the mid gut was measured in the zven1 treated animals overvehicle control (37.1% compared to 15% (p=0.004). At the final, 775ug/kg dose, increased gastric emptying was detected over control 46.6%compared to 63.8%, but the effect was not as great as the 77.5 μg/kgdose. Increased intestinal transit was detected in the mid gut (26%versus 15%), but the effect was not as significant as that observed withthe lower 77.5 ug/kg dose. These data suggest that at higherconcentrations, zven1 can inhibit gastric emptying and intestinaltransport.

TABLE 7 Description of treatment groups and results Number of % TestMeal % Test Meal in % Test Meal Treatment Groups Animals in StomachProximal Gut in Mid Gut Group I Vehicle (Buffer N = 10 63.8% ± 3.8%18.4% ± 2.4%   15% ± 3.3% SE for zven1) SE SE Group II zven1 0.78 μg/kgN = 10 56.3% ± 5.2% 25.5% ± 4.1% 14.6% ± 4% SE body weight SE SE *GroupIII zven1 7.8 μg/kg N = 10 43.7% ± 3.2% 35.0% ± 5.4% 18.5% ± 5.1% bodyweight SE SE SE *p = .001 *p = .004 *Group IV zven1 N-10 37.8% ± 4.5%26.6% ± 5.1% 37.1% ± 7.1% 77.5 μg/kg body weight SE SE SE *p = .0002 *p= .004 *Group V zven1 775 μg/kg N = 10 46.6% ± 4.5% 24.0% ± 5.9%   26% ±4.3% SE body weight SE SE *p = .05 *p = .009 Group VI Caerulein (CCK N =10 93.8% ± 1.0%  2.6% ± 0.9%  1.2% ± 0.3% analogue positive control) SESE SE 40 ng/g body weight Group VII Sham non- N = 5 100% NA NA treated

Example 9 Baculovirus Expression of Zven1

An expression vector containing a GLU-GLU tag, pzBV32L:zven1cee, wasdesigned and prepared to express zven1cee polypeptides in insect cells.

A. Expression Vector:

An expression vector, pzBV32L:zven1cee, was prepared to express humanzven1 polypeptides having a carboxy-terminal Glu-Glu tag, in insectcells as follows.

A 371 bp fragment containing sequence for zven1 and a polynucleotidesequence encoding EcoR1 and Xba1 restriction sites on the 5′ and 3′ends, respectively, was generated by PCR amplification using PCRSuperMix (Gibco BRL, Life Technologies) and appropriate buffer from aplasmid containing zven1 cDNA (zven1-zyt-1.contig) using primers ZC29463(SEQ ID NO:23) and ZC29462 (SEQ ID NO:24). (Note: the zven1 sequence andthe Xba1 site was out of frame. An additional 2 bases, CC—antisense,were added to put in frame, which coded for an additional Gly betweenthe zven1 sequence and the CEE tag.) The PCR reaction conditions were asfollows: 1 cycle of 94° C. for 3 minutes, followed by 25 cycles of 94°C. for 30 seconds, 50° C. for 30 seconds, and 68° C. for 30 seconds;followed by a 4° C. hold. The fragment was visualized by gelelectrophoresis (1% Agarose-1 μl of 10 mg/ml EtBr per 10 ml of agarose).A portion of the PCR product was digested with EcoR1 and Xba1restriction enzymes in appropriate buffer, then run on an agarose gel.DNA corresponding to the EcoR1/Xba1 digested zven1 coding sequence wasexcised, purified using Qiagen Gel Extraction kit (#28704), and ligatedinto an EcoR1/XbaI digested baculovirus expression donor vector,pZBV32L. The pZBV32L vector is a modification of the pFastBac1™ (LifeTechnologies) expression vector, where the polyhedron promoter has beenremoved and replaced with the late activating Basic Protein Promoter. Inaddition, the coding sequence for the Glu-Glu tag (SEQ ID NO:10) as wellas a stop signal is inserted at the 3′ end of the multiple cloningregion. About 216 nanograms of the restriction digested zven1 insert andabout 300 ng of the corresponding vector were ligated overnight at 15°C. One μl of ligation mix was electroporated into 35 μl DH10B cells(Life Technologies) at 2.1 kV. The electroporated DNA and cells werediluted in 1 ml of LB media, grown for 1 hr at 37° C., and plated ontoLB plates containing 100 μg/ml ampicillin. Clones were analyzed byrestriction digests and one positive clone was selected and streaked onAMP+ plates to get single colonies for confirmation by sequencing.

Sequencing revealed the presence of a initiation codon upstream of theactual start codon which would possibly interfere with propertranslation. Therefore, the upstream codon was removed using aQuick-change mutagenesis kit from Stratagene (La Jolla, Calif.). Thiswas accomplished by designing forward and reverse primers that changedthe upstream initiation ATG to a ATC, thereby also eliminating a Ncorestriction digest site and creating a Sma1 site instead. The newmutagenized plasmid containing the Sma1 and Xba1 cleavage sites at the5′ and 3′ ends of the zven1 sequence was then electroporated into DH10Bcells as before, analyzed by restriction digests, this time with Sma1and Xba1, and a positive clone was selected and streaked on AMP+ platesto get a single colony for confirmation by sequencing as before. A clonefor the Zven1 polynucleotide sequence could also be cloned without theupstream initiation codon.

One to 5 ng of the positive clone donor vector was transformed into 100μl DH10Bac Max Efficiency competent cells (GIBCO-BRL, Gaithersburg, Md.)according to manufacturer's instruction, by heat shock for 45 seconds ina 42° C. waterbath. The transformed cells were then diluted in 980 μlSOC media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl,1.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄ and 20 mM glucose) out-grown inshaking incubator at 37° C. for four hours and plated onto Luria Agarplates containing 50 μg/ml kanamycin, 7 μg/ml gentamicin, 10 μg/mltetracycline, IPTG and Blue Gal. The plated cells were incubated for 48hours at 37° C. A color selection was used to identify those cellshaving zVen1cee encoding donor insert that had incorporated into theplasmid (referred to as a “bacmid”). Those colonies, which were white incolor, were picked for analysis. Bacmid DNA was isolated from positivecolonies using standard isolation technique according to LifeTechnologies directions. Clones were screened for the correct insert byamplifying DNA using primers to the transposable element in the bacmidvia PCR. The PCR reaction conditions were as follows: 35 cycles of 94°C. for 45 seconds, 50° C. for 45 seconds, and 72° C. for 5 minutes; 1cycle at 72° C. for 10 min.; followed by 4° C. soak. The PCR product wasrun on a 1% agarose gel to check the insert size. Those having thecorrect insert size were used to transfect Spodoptera frugiperda (Sf9)cells. The polynucleotide sequence is shown in SEQ ID NO:25. Thecorresponding amino acid sequence is shown in SEQ ID NO:26.

B. Transfection in Insect Cells:

Sf9 cells were seeded at 1×10⁶ cells per 35 mm plate and allowed toattach for 1 hour at 27° C. Five micrograms of bacmid DNA was dilutedwith 100 PI Sf-900 II SFM medium (Life Technologies, Rockville, Md.).Fifteen μl of lipofectamine Reagent (Life Technologies) was diluted with100 μl Sf-900 II SFM. The bacmid DNA and lipid solutions were gentlymixed and incubated 30-45 minutes at room temperature. The media fromone plate of cells was aspirated. Eight hundred microliters of Sf-900 IISFM was added to the lipid-DNA mixture. The DNA-lipid mix was added tothe cells. The cells were incubated at 27° C. overnight. The DNA-lipidmix was aspirated the following morning and 2 ml of Sf-900 II media wasadded to each plate. The plates were incubated at 27° C., 90% humidity,for 168 hours after which the virus was harvested.

C. Primary Amplification

Sf9 cells were seeded at 1×10⁶ cells per 35 mm plate and allowed toattach for 1 hour at 27° C. They were then infected with 500 μl of theviral stock from above and incubated at 27° C. for 4 days after whichtime the virus was harvested according to standard methods known in theart.

D. Secondary Amplification

Sf9 cells were seeded at 1×10⁶ cells per 35 mm plate and allowed toattach for 1 hour at 27° C. They were then infected with 20 μl of theviral stock from above and incubated at 27° C. for 4 days after whichtime the virus was harvested according to standard methods known in theart.

E. Tertiary Amplification

Sf9 cells were grown in 80 ml Sf-900 II SFM in 250 ml shake flask to anapproximate density of 1×10⁶ cells/ml. They were then infected with 200μl of the viral stock from above and incubated at 27° C. for 4 daysafter which time the virus was harvested according to standard methodsknown in the art.

F. Expression of Zven1Cee

Third round viral stock was titered by a growth inhibition curve and theculture showing an MOI of “1” was allowed to proceed for 48 hrs. Thesupernatant was analyzed via Western blot using a primary monoclonalantibody specific for the n-terminal Glu Glu epitope and a HRPconjugated Gt anti Mu secondary antibody. Results indicated a band ofthe predicted molecular weight.

A large viral stock was then generated by the following method: Sf9cells were grown in IL Sf-900 II SFM in a 2800 ml shake flask to anapproximate density of 1×10⁶ cells/ml. They were then infected withviral stock from the 3^(rd) round amp. and incubated at 27° C. for 72hrs after which time the virus was harvested. Larger scale infectionswere completed to provide material for downstream purification.

Example 10 Expression in E. coli

A. Generation of the Native Zven1 Expression Construct

A DNA fragment of native Zven1 (SEQ ID NO:11) was isolated using PCR.Primer zc #40,821 (SEQ ID NO:12) containing 41 bp of vector flankingsequence and 24 bp corresponding to the amino terminus of Zven1, andprimer zc#40,813 (SEQ ID NO:13) contained 38 bp corresponding to the 3′end of the vector which contained the zven1 insert. Template waspZBV32L:zven1cee. The PCR conditions were as follows: 25 cycles of 94°C. for 30 seconds, 50° C. for 30 seconds, and 72° C. for 1 minute;followed by a 4° C. soak. A small sample (2-4 μL) of the PCR sample wasrun on a 1% agarose gel with 1×TBE buffer for analysis, and the expectedband of approximately 500 bp fragment was seen. The remaining volume ofthe 100 μL reaction was precipitated with 200 μL absolute ethanol.Pellet was resuspended in 10 μL water to be used for recombining intoSma1 cut recipient vector pTAP238 to produce the construct encoding thezven1 as disclosed above. The clone with correct sequence was designatedas pTAP432. It was digested with Not1/Nco1 (10 μl DNA, 5 μl buffer 3 NewEngland BioLabs, 2 μL Not 1, 2 μL Nco 1, 31 μL water for 1 hour at 37°C.) and religated with T4 DNA ligase buffer (7 μL of the previousdigest, 2 μL of 5× buffer, 1 μL of T4 DNA ligase). This step removed theyeast sequence, CEN-ARS, to streamline the vector. The DNA wasdiagnostically digested with Pvu 2 and Pst 1 to confirm the absence ofthe yeast sequence. DNA was transformed into E. coli strain W3110/pRARE.

B. Expression of the Native Zven1 in E. coli

E. coli was inoculated into 100 ml Superbroth II medium (BectonDickinson, Franklin Lakes, N.J.) with 0.01% Antifoam 289 (Sigma), 30μg/ml kanamycin, 35 μg/ml chloramphenicol and cultured overnight at 37°C. A 5 ml inoculum was added to 500 ml of the same medium in a 2 Lculture flask which was shaken at 250 rpm at 37° C. until the cultureattained an OD₆₀₀ of 4. IPTG was then added to a final concentration of1 mM and shaking was continued for another 2.5 hours. The cells werecentrifuged at 4,000×g for 10 min at 4° C. The cell pellets were frozenat −80° C.

Example 11 Codon Optimization

A. Generation of the Codon Optimized Zven1 Expression Construct

Native human Zven1 gene sequence could not be expressed in E. colistrain W3110. Examination of the codons used in the Zven1 codingsequence indicated that it contained an excess of the least frequentlyused codons in E. coli with a CAI value equal to 0.211. The CAI is astatistical measure of synonymous codon bias and can be used to predictthe level of protein production (Sharp et al., Nucleic Acids Res.15(3):1281-95, 1987). Genes coding for highly expressed proteins tend tohave high CAI values (>0.6), while proteins encoded by genes with lowCAI values (≦0.2) are generally inefficiently expressed. This suggesteda reason for the poor production of Zven1 in E. coli. Additionally, therare codons are clustered in the second half of the message leading tohigher probability of translational stalling, premature termination oftranslation, and amino acid misincorporation (Kane J F. Curr. Opin.Biotechnol. 6(5):494-500, 1995).

It has been shown that the expression level of proteins whose genescontain rare codons can be dramatically improved when the level ofcertain rare tRNAs is increased within the host (Zdanovsky et al.,ibid., 2000; Calderone et al., ibid., 1996; Kleber-Janke et al., ibid.,2000; You et al., ibid., 1999). The pRARE plasmid carries genes encodingthe tRNAs for several codons that are rarely used E. coli (argU, argW,leuW, proL, ileX and glyT). The genes are under the control of theirnative promoters. Co-expression with pRARE enhanced Zven1 production inE. coli and yielded approximately 100 mg/L. Co-expression with pRAREalso decreased the level of truncated zven1 in E. coli lysate. Thesedata suggest that re-resynthesizing the gene coding for zven1 with moreappropriate codon usage provides an improved vector for expression oflarge amounts of zven1.

The codon optimized zven1 coding sequence (SEQ ID NO:14) was constructedfrom six overlaping oligonucleotides: zc45,048 (SEQ ID NO:15), zc45,049(SEQ ID NO:16), zc45,050 (SEQ ID NO:17), zc45,051 (SEQ ID NO:18),zc45,052 (SEQ ID NO:19) and zc45,053 (SEQ ID NO:20). Primer extension ofthese overlapping oligonucleotides followed by PCR amplificationproduced a full length zven1 gene with codons optimized for expressionin E. coli. The final PCR product was inserted into expression vectorpTAP237 by yeast homologous recombination. The expression construct wasextracted from yeast and transformed into competent E. coli DH10B.Clones resistance to kanamycin were identified by colony PCR. A positiveclone was verified by sequencing and subsequently transformed intoproduction host strain W3110. The expression vector with the optimizedzven1 sequence was named pSDH187. The resulting gene was expressed verywell in E. coli. Expression levels with the new construct increased toaround 150 mg/L.

B. Expression of the Codon Optimized Zven1 in E. coli

E. coli was inoculated into 100 ml Superbroth II medium (BectonDickinson) with 0.01% Antifoam 289 (Sigma), 30 μg/ml kanamycin andcultured overnight at 37° C. A 5 ml inoculum was added to 500 ml of samemedium in a 2 L culture flask which was shaken at 250 rpm at 37° C.until the culture attained an OD₆₀₀ of 4. IPTG was then added to a finalconcentration of 1 mM and shaking was continued for another 2.5 hours.The cells were centrifuged at 4,000×g for 10 min at 4° C. The cellpellets were frozen at −80° C. until use at a later time.

Example 12 Purification and Refolding of Zven1 Produced in E. coli

A. Inclusion Body Isolation:

Following induction of protein expression in either batch ferment orshaker flask culture, the E. coli broth was centrifuged in 1 literbottles at 3000 RPM in a Sorvall swinging bucket rotor. Additionalwashing of the cell paste to remove any broth contaminants was performedwith 50 mM Tris pH 8.0 containing 200 mM NaCl and 5 mM EDTA until thesupernate was clear.

The cell pellets were then suspended in ice cold lysis buffer (50 mMTris pH 8.0; 5 mM EDTA; 200 mM NaCl, 10% sucrose (w/v); 5 mM DTT; 5 mMBenzamidine;) to 10-20 Optical Density units at 600 nm. This slurry wasthen subjected to 2-3 passes at 8500-9000 psi in a chilled APV 2000 LabHomogenizer producing a disrupted cell lysate. The insoluble fraction(inclusion bodies) was recovered by centrifugation of the cell lysate at20,000×G for 1 hour at 4° C.

The inclusion body pellet (resulting from the 20,000×G spin) wasre-suspended in wash buffer (50 mM Tris pH 8 containing 200 mM NaCl, 5mM EDTA, 5 mM DTT, 5 mM Benzamidine) at 10 ml wash buffer per graminclusion bodies, and was completely dispersed utilizing an OMNIinternational rotor stator generator. This suspension was centrifuged at20,000×G for 30 minutes at 4° C. The wash cycle was repeated 3-5 timesuntil the supernatant was clear.

The final washed pellet was solubilized in 8M Urea, 50 mM Borate bufferat pH 8.6 containing 0.1M Sodium Sulfite and 0.05 M Sodium Tetrathionateat pH 8.2. The solubilization and sulfitolysis reaction was allowed toproceed at 4° C. overnight with gentle shaking. The resulting pinkishcolored solution was centrifuged at 35,000×g for 1 hour at 4° C. and theclarified supernate, containing the soluble Zven1, was 0.45 um filtered.

B. Zven1 Refolding:

The solubilized Zven1 was refolded by drop-wise dilution into ice coldrefolding buffer containing 55 mM Borate pH 8.6, 1.0 M Arginine, 0.55 MGuanidine HCL, 10.56 mM NaCl, 0.44 mM KCl, 0.055% PEG, 10 mM reducedGlutathione and 1.0 mM oxidized Glutathione at a final zven1concentration of 100-150 ug/ml. Once diluted, the mixture was allowed tostir slowly in the cold room for 48-72 hours.

C. Product Recovery & Purification:

After refolding, the solution was clarified by centrifugation at22,000×G, 1 hour, 4° C. and/or by filtration using a 0.45 micronmembrane. The clarified supernate, containing refolded zven1, wasadjusted to 50 mM acetate and the pH adjusted to 4.5 with addition ofHCl. The pH adjusted material was captured by cation exchangechromatography on a Pharmacia Streamline SP column (33 mm ID×65 mmlength) equilibrated in 50 mM acetate pH 4.5 buffer. The load flow ratewas 10 ml/min with inline dilution proportioning 1:5 in 50 mM acetatebuffer at pH 4.5. This dilution lowers the ionic strength enablingefficient binding of the target to this matrix. After sample loading wascomplete, the column was washed to baseline absorbance withequilibration buffer prior to step elution with 50 mM acetate pH 4.5buffer containing 1 M NaCl.

The eluate pool from the cation exchange step was brought to 1% Aceticacid, pH 3.0 and Loaded to a column (22 mm×130 mm) containing Toso HassAmberchrom CG71m reverse phase media equilibrated in 1% acetic acid, pH3.0 at a flow rate of 10 ml/min. Upon washing to baseline absorbance,the column was eluted with a 20 column volume gradient formed betweenequilibration buffer and 99% (V/V) acetonitrile, 1% (V/V) acetic acid.

The eluate pool from the reverse phase step was subjected to anotherround of cation exchange chromatography. The pool was directly loaded onto a Toso Haas SP 650 S column (10 mm×50 mm) equilibrated in 50 mMacetete pH 4.5 buffer at a flow rate of 3 ml/min. Upon completing thesample load, and washing to baseline absorbance, the column was stepeluted with 50 mM acetate pH 3.0 buffer containing 1.0 M NaCl. Theprotein eluate pool was concentrated against a 3 k Da cutoffultrafiltration membrane using an Amicon concentration unit inpreparation for the final purification and buffer exchange sizeexclusion step.

D. Size Exclusion Buffer Exchange and Formulation:

The concentrated cation pool was injected onto a Pharmacia SuperdexPeptide size exclusion column (Pharmacia, now Pfizer, La Jolla, Calif.)equilibrated in 25 mM Histidine; 120 mM NaCl at pH 6.5. The symetriceluate peak containing the product was pooled, 0.2 micronsterile-filtered, aliquoted and stored at −80° C.

Example 13 Activity of Zven1 and Zven2 in a Reporter Assay

A. Cell Lines

Rat2 fibroblast cells (ATCC #CRL-1764, American Type Culture Collection,Manassass, Va.) were transfected with a SRE luciferase reporterconstruct and selected for stable clones. These were then transfectedwith constructs for either GPCR73a receptor (SEQ ID NO:21) or GPCR73breceptor (SEQ ID NO:22).

B. Assay Procedure

Cells were trypsinized and seeded in Corning 96-well white plates at3,000 cells/well in media containing 1% serum and incubated overnight at37° C. and 5% CO₂. Media was removed and samples were added intriplicate to cells in media containing 0.5% BSA and incubated for fourhours at 37° C. and 5% CO₂. After media was removed the cells were lysedand luciferase substrate was added according to the Promega luciferaseassay system (Promega Corp., Madison, Wis.)

C. Data and Conclusions

All data were reported as fold-induction of the RLU (relative lightunits) from the luminometer divided by the basal signal (media only).Zven1 was prepared in house. Zven2 used in the assay was purchased fromPeproTech Inc. (Rocky Hill, N.J.).

Tables 8 and 9 show that Zven1 was more active than Zven2 in adose-dependent manner with cells expressing the GPCR73a receptor.

TABLE 8 GPCR 73a Fold-induction conc. (ng/ml) Zven1 (E. coli produced)Zven2 1000 17.8 20 320 20.7 24.4 100 19 11.4 32 15 5.8 10 8.4 2.5 3.2 41.6 1 1.9 1.2

TABLE 9 GPCR73a Fold-induction conc. (ng/ml) Zven1 (E. coli produced)Zven2 1000 13.9 15 320 22 20.5 100 17.6 11.4 32 14.1 7.2 10 10.2 2.6 3.27.6 1.3 1 4.1 0.95Table 10 and 11 show that Zven1 and Zven2 were similar in activity withthe cells expressing the GPCR73b receptor. Activity of both moleculeswas lower in the cells expressing the GPCR73b receptor. It is not knownif the GPCR73b receptor numbers were equivalent in both cell lines.

TABLE 10 GPCR73b Fold-induction conc. (ng/ml) Zven1 (E. coli produced)Zven2 1000 7.1 8.4 320 6.3 8.3 100 4.7 5.6 32 3 2.8 10 1.9 1.8 3.2 1.31.3 1 0.7 1.1

TABLE 11 GPCR73b Fold-induction conc. (ng/ml) Zven1 (E. coli produced)Zven2 1000 4.8 6.1 320 5.2 5.8 100 4.4 4.1 32 2.6 2.7 10 1.7 1.8 3.2 1.21.4 1 1 1.1Table 12 shows that Baculovirus-expressed Zven1 that has been heated at56° C. for 30 minutes may have reduced activity than fresh Zven1.

TABLE 12 GPCR73a Fold-induction conc. (ng/ml) Fresh Zven1 Heated Zven1100 20.5 18.6 32 18.7 14.8 10 13.1 10 3.2 7.1 3.7 1 2.5 1.8

Example 14 Zven1 Activity in Organ Bath

Organ bath testing was also performed with Zven1 using at a variety oftissues obtained from guinea pigs. A force transducer was used to recordthe mechanical contraction using IOX software (EMKa technologies, FallsChurch, Va.) and Datanalyst software (EMKa technologies, Falls Church,Va.). Tissues analyzed included: duodenum, jejunum, ileum, trachea,esophagus, aorta, stomach, gall bladder, bladder and uterus.

A. Organ Bath Methods

Two month old male guinea pigs (Hartley, Charles River Labs) weighing˜250 to 300 g were fasted with access to drinking water for ˜18 hoursthen euthanized by CO₂ asphyxiation. All tissues were rinsed with Krebsbuffer (1.2 mM MgSO₄, 115 mM NaCl, 11.5 mM glucose, 23.4 mM NaHCO₃, 4.7mM KCl, 1.2 mM NaH₂PO₄, and 2.4 mM CaCl₂, oxygenated with 95% O₂-5% CO₂,pH 7.4, temperature 37° C.) then suspended in the 5 ml organ bath andpre-tensioned. All tissues were tested with positive controls toestablish their viability prior to running. Positive controls used wereCCK-8, acetylcholine (ACH), histamine, or 5HT, and were purchased fromSigma (Saint Louis, Mo.). All tissues were treated with a vehiclecontrol, phosphate buffered saline (PBS), to rule out the possibility ofvehicle effects.

1) Tissues that Did not Give a Response to Zven1 in the Organ Bath:

-   -   Tracheal ring: 3 mm wide tracheal ring (3 cm away from brachial        branches) was collected and allowed to equilibrate at 5 gram        tension prior to any treatments. The positive control was 20        ug/ml ACH, which gave an approximate 1 gram deflection. No        effect seen with Zven1 at 80 ng/ml.    -   Aortic ring: 3 mm wide aortic ring (immediately adjacent to        aortic arch) was collected and allowed to equilibrate at 4 gram        tension prior to any treatments. The positive control was 2        mg/ml KCl, which gave an average one gram deflection. Zven1 at        80 ng/ml did not cause a visible effect.    -   Esophagus: 2 cm in length esophagus (2 cm away from cardia) was        suspended and allowed to equilibrate at 1 gram tension prior to        any treatments. Two mg/ml 5HT gave an approximate 1.4 grams        deflection. Zven1 at 20 ng/ml had no visible effect.    -   Gall bladder: Lumenal fluid was aspirated out with 1 ml syringe        then longitudinally suspended and allowed to equilibrate at 1        gram tension prior to any treatments. Five ng/ml of ACH gave a        0.4 gram deflection response. No effect was seen with 20 ng/ml        zven1.    -   Bladder: 1.5 cm×0.3 cm longitudinal strip was suspended and        allowed to equilibrate to 0.5 gram tension prior to any        treatments. Positive controls induced a contractile response,        but no activity was seen at a 80 ng/ml Zven1 dose.

2) Tissues that Responded to Zven1:

-   -   Stomach/antrum: 1.5 cm×0.3 cm longitudinal strip was suspended        and allowed to equilibrate to 0.5 gram tension prior to any        treatments. Treatment with either 5 ng/ml ACH or 80 ng/ml CCK 8        resulted in an approximate one gram deflection. Eighty ng/ml        Zven1 also produced a contractile response of approximately 0.5        gm deflection.    -   Duodenum: 2 cm in length duodenum (2 cm away from pylorus) was        suspended and allowed to equilibrate at 1 gram tension prior to        any treatments. ACH gave an approximate 0.75 gm deflection.        Twenty ng/ml Zven1 also gave a contractile response of        approximately 0.5 grams deflection.    -   Jejunum: 2 cm in length jejunum (midpoint between pylorus and        ileal-cecal junction) was suspended and allowed to equilibrate        at 1 gram tension prior to any treatments. ACH gave an        approximate 1.0 gram deflection and 20 ng/ml Zven1 gave an        approximate 0.5 gram deflection contractile response.    -   Ileum: 8 cm in length ileum (2 cm away from ileal-cecal        junction) was collected and flushed with Krebs buffer to remove        any fecal debris if present then cut into four equal pieces. All        tissues were suspended and allowed to equilibrate at 1 gram        tension prior to any treatments. The ileum was run at the same        time to compare Zven1 effects on the small intestine. ACH gave        an approximate 1.5 gram deflection, and 20 ng/ml Zven1 also gave        a 1.5 gram deflection.    -   Proximal Colon: 2 cm in length colon (2 cm away from cecum) was        suspended and allowed to equilibrate at 0.5 gram tension prior        to any treatments. Zven1 at 20 ng/ml induced a relaxation effect        with a decrease in muscle tone and a decrease in the amplitude        of the contractions.

Zven1's contractile effects are specific to the gastrointestinal tract.The greatest contractile response is seen in the ileum, with lessercontraction seen in the duodenum, jejunum, and antrum. The relaxationeffect in the proximal colon is suggestive of a coordinated effect ongut motility. As the smooth muscle contraction is enhanced in the antrumand the small intestine, the large intestine is preparing to accommodatethe approaching meal by relaxing. Coordinated contractile activitybetween different parts of the gut will result in improvedgastrointestinal function.

Example 15 Comparative Activity of Zven1 and Zven2 in the Organ Bath

Both Zven1 and Zven2 have contractile effects on intestinal tissue inthe organ bath. Side by side comparisons were made to compare activityin tissue derived from the same animal.

Ileal strips from guinea pig were tested for contractility using methodsdescribed above. Zven2 was purchased from PeproTech Inc. (Rocky Hill,N.J.). Activity was compared at 40, 12, and 3 ng/ml concentrations. ACHat 5 ng/ml was used as a positive control. Contractile responses werenormalized to the ACH response in each tissue. All three doses were runon separate ileal longitudinal tissue strips obtained from the sameanimal.

Results: Contractile effects were normalized to the ACH positive controland are expressed as the ratio of Zven1 or Zven2 to ACH in the tablebelow.

TABLE 13 Conc Zven1 Zven2 (ng/ml) ACH Zven1 Zven1:ACH ACH Zven2Zven2:ACH 40 1.26 1.28 1.02 1.25 0.58 0.46 12 2.5 2.51 1.00 2.26 0.61.027 3 1.38 .047 .034 1.73 .027 .016

Conclusions: Zven1 is approximately twice as active as Zven2 whencomparing contractility in the ileum.

Example 16 Synergistic Effects of Zven1 and Zven2

In order to determine the combined effects of Zven1 and zven2 oncontractile activity, ileal tissues were pre-treated with varying dosesof zven2, followed by increasing doses of zven1.

All tissues are stabilized, treated with ACH, and again stabilized priorto pre-treatment with zven2 at concentrations of 0.8, 3.0 or 12 ng/ml.Zven2 was left on tissue for approximately 20 minutes prior to dosingwith 20 ng/ml zven1.

Results: Large 3 gram deflection contractions with Zven1 were observedwhen the tissue was pre-treated with 0.8 ng/ml zven2. These contractionswere larger than what is normally observed with a 20 ng/ml dose ofzven1, where contractile effects of approximately 1.5 to 2.0 gramsdeflection are normally observed. Zven2 alone at 0.8 ng/ml has anegligible contractile effect.

Conclusions: These data suggest that by pre-treating with a low dose ofzven2, and then treating with zven1, increased motility effects may beobtained.

Example 17 MIP-2 Detection in Lavage Fluids and Serum of Mice FollowingIP (Intraperitoneal) Injection of Zven1

As discussed in Example 3, above, mouse KC is the mouse homolog of humanGROα, and CINC-1 is the rat homolog. Similarly, increased MIP-2expression has been found to be associated with neutrophil influx invarious inflammatory conditions. See Banks, C. et al, J. Path. 199:28-35, 2003.

Similar to the methods used in Example 3, four groups of ten mice wereinjected with Zven1 at 5 and 50 ug/kg, a vehicle control, or notreatment. These mice weighed approximately 20 grams, so the dose was 5μg/kg. MIP-2 levels were measured in both peritoneal lavage fluid andserum using a Quantikine M Murine mouse MIP-2 ELISA kit (R and DSystems, Minneapolis, Minn.). Test results are shown in Table 14.

TABLE 14 MIP-2 picograms/ml Serum Lavage Fluid Non-treated control 6.2+/− 1.3 5.9 +/− 0.7 Vehicle 6.7 +/− 1.3 16.7 +/− 2.2  5 ug/kg Zven1 14.3+/− 2.7  21.5 +/− 3.7  50 ug/kg Zven1 7.7 +/− 1.8 8.7 +/− 1.2 Data =mean +/− SEM

Conclusions: MIP-2 is up-regulated in serum and lavage fluid in responseto a low, (5 ug/kg), IP injection of zven1. Concentrations in serum areapproximately 2-fold higher in the zven1 treated animals. There is alesser effect in lavage fluid, but that is due to the fact that someactivation took place in the vehicle treated animals over non-treatedcontrol animals. At the higher (50 ug/kg dose) no effect was observedsuggesting that at elevated doses there is no chemotactic effect. Theseresults correlate with the neutrophil numbers, where in, neutrophilinfiltration was observed only in the animals administered the lower (5ug/kg) dose of Zven1.

Example 18 Production of Zven1 Polyclonal Antibodies

Polyclonal antibodies were prepared by immunizing 2 female New Zealandwhite rabbits with the purified recombinant protein huzven1-CEE-Bv (SEQID NO:24) The rabbits were each given an initial intraperitoneal (ip)injection of 200 μg of purified protein in Complete Freund's Adjuvantfollowed by booster ip injections of 100 μg peptide in IncompleteFreund's Adjuvant every three weeks. Seven to ten days after theadministration of the second booster injection (3 total injections), theanimals were bled and the serum was collected. The animals were thenboosted and bled every three weeks.

Polyclonal antibodies were purified from the immunized rabbit serumusing a 5 ml Protein A sepharose column (Pharmacia LKB). Followingpurification, the polyclonal antibodies were dialyzed with 4 changes of20 times the antibody volume of PBS over a time period of at least 8hours. Huzven1-specific antibodies were characterized by ELISA using 500ng/ml of the purified recombinant protein huzven1-CEE-Bv (SEQ ID NO:24)as the antibody target. The lower limit of detection (LLD) of the rabbitanti-huzven1 purified antibody was 1 ng/ml on its specific purifiedrecombinant antigen huzven1-CEE-Bv.

Example 19 Detection of Zven1 Protein

The purified polyclonal huzven1 antibodies were characterized for theirability to bind recombinant human Zven1 polypeptides using the ORIGEN®Immunoassay System (IGEN Inc, Gaithersburg, Md.). In this assay, theantibodies were used to quantitatively determine the level ofrecombinant huzven1 in rat serum samples. An immunoassay format wasdesigned that consisted of a biotinylated capture antibody and adetector antibody, which was labeled with ruthenium (II) tris-bipyridalchelate, thereby sandwiching the antigen in solution and forming animmunocomplex. Streptavidin-coated paramagnetic beads were then bound tothe immunocomplex. In the presence of tripropylamine, the ruthenylatedAb gave off light, which was measured by the ORIGEN analyzer.Concentration curves of 0.1-50 ng/ml huzven1 made quantitation possibleusing 50 microliters of sample. The resulting assay exhibited a lowerlimit of detection of 200 pg/ml huzven1 in 5% normal rat serum.

Example 20 Effect of Zven1 in Post-Operative Ileus In Vivo

Five to 25 male Sprague-Dawley rats (˜240 g) per treatment group wereused for these POI studies. Animals were fasted for ˜22-23 h (with 2floor grids placed in their cages to prevent them from having access totheir bedding) with free access to water. While under gas isofluraneanesthesia, the rat's abdomen was shaved and wiped with betadine/70%ethanol. A midline incision was then made through the skin and lineaalba of the abdomen (3-4 cm long), such that intestines were visible andaccessible. The cecum was manipulated for 1 min with sterilesaline-soaked gauze, using a gentle, pulsatile-like pressure. Thisprocedure was consistent from animal to animal in order to reduceinter-animal ileus variability. The linea alba was sutured with silksuture and the skin closed with wound clips. Animals were kept onwater-jacketed heating pads during recovery from surgery and placed backinto their cages once they regained full consciousness.

When fully conscious, rats were administered 1.0 ml of the test meal 15minutes following completion of cecal manipulation (CM); one minute or20 minutes later, rats were administered 0.8 or 5 ug/kg BW E.coli-produced Zven1, or saline/0.1% w/v/BSA via indwelling jugularvenous catheter. Zven1 was diluted with saline/0.1% BSA to the desiredconcentration (based on average BW of rat [˜240 g] and a 0.1 mlinjection volume for i.v.) immediately prior to study, using siliconizedmicrofuge tubes.

The test meal consisted of 1.5% (w/v) aqueous methylcellulose solution(medium viscosity methylcellulose from Sigma 400 centipoises; catalog #M-0262) along with a non-absorbable dye, 0.05% (50 mg/100 ml) phenol red(Sigma catalog # P-4758; lot #120K3660). Twenty minutes followingadministration of the test meal, animals were anesthetized underisoflurane and sacrificed by cervical dislocation. The stomach andintestinal segments were removed, and the amount of phenol red in eachsegment was determined by spectrophotometric analysis (see below) andexpressed as the percent of total phenol red recovered per rat. Thesevalues are used to determine the amount of gastric emptying and guttransit per tissue collected.

Colorimetric analysis of phenol red recovered from each gut segment andstomach were performed according to a modification of the procedureoutlined by Scarpinato and Bertaccini (1980) and Izbeki et al (2002).Briefly, following euthanization, the stomach and intestinal segmentswere placed into 20 ml of 0.1 N NaOH and homogenized using a Polytrontissue homogenizer. The Polytron was then rinsed with 5 ml of 0.1 N NaOHand added to the previous 20 ml, along with another 15 ml of 0.1 N NaOH.Homogenate was allowed to settle for at least 1 hour at roomtemperature. Proteins were precipitated from 5 ml of the supernate bythe addition of 0.5 ml of 20% trichloracetic acid. Followingcentrifugation (3000 rpm for 15 min), 1 ml of supernatant was added to 1ml of 0.5 N NaOH. A 0.2 ml sample (in a 96-well plate) was read at 560nm using Molecular Devices Spectra Max 190 spectrophotometer. The extentof gastric emptying and intestinal transit were expressed as percent oftotal phenol red recovered per rat.

Data indicated that Zven1 (0.8 and 5.0 ug/kg, i.v.) significantlyincreased gastric emptying and upper intestinal transit of thissemi-solid, non-nutritive meal by approximately 1.6 to 2.0-fold comparedto emptying and transit observed in vehicle-treated rats. Efficacy inthis model was observed when these doses of Zven1 are administered ateither 1 min or 20 min following meal administration.

Example 21 Effect of i.v. and ip. BV- and E. Coli-Produced Zven1 onGastric Emptying and Intestinal Transit of a Phenol Red Semi-Solid Mealin Rats

Male Sprague-Dawley rats (˜240 g) were used for this study, with 6-12animals per treatment group. Animals were fasted for ˜24 h (with 2 floorgrids placed in their cages to prevent them from having access to theirbedding) with free access to water. One minute following theadministration of 1.0 ml of test meal, rats were administered varyingdoses of Zven1 (0.01 to 30 ug/kg BW) or saline/0.1% w/v BSA viaindwelling jugular venous catheter. For i.p. dosing, Zven1 (0.1 to 100ug/kg BW) or saline/0.1% BSA was administered either 1 or 10 min priorto or 1 min after the meal. Zven1 was diluted with saline/0.1% BSA tothe desired concentration (based on average BW of rat [˜240 g] and a 0.1ml injection volume for i.v. or 0.5 ml injection volume for i.p.)immediately prior to study, using siliconized microfuge tubes. The testmeal consisted of 1.5% (w/v) aqueous methylcellulose solution (mediumviscosity methylcellulose from Sigma 400 centipoises; catalog # M-0262)along with a non-absorbable dye, 0.05% (50 mg/100 ml) phenol red (Sigmacatalog # P-4758; lot #120K3660). Fifteen or 20 min followingadministration of the test meal, rats were anesthetized under isofluraneand sacrificed by cervical dislocation.

The stomach and intestinal segments were removed, and the amount ofphenol red in each sample was determined by spectrophotometric analysis(see below) and expressed as the percent of total phenol red recoveredper rat. These values were used to determine the amount of gastricemptying and gut transit per tissue collected.

Colorimetric analysis of phenol red recovered from each gut segment andstomach were performed according to a modification of the procedureoutlined by Scarpinato et al Arch Int. Pharmacodyn. 246:286-294 (1980)and Piccinelli et al. Naunyn-Schmiedeberg's Arch. Pharmacol 279: 75-82(1973). Briefly, following euthanization, the stomach and intestinalsegments were placed into 20 ml of 0.1 N NaOH and homogenized using aPolytron tissue homogenizer. The Polytron was then rinsed with 5 ml of0.1 N NaOH and added to the previous 20 ml, along with another 15 ml of0.1 N NaOH. Homogenate was allowed to settle for at least 1 hour at roomtemperature. Proteins were precipitated from 5 ml of the supernate bythe addition of 0.5 ml of 20% trichloracetic acid. Followingcentrifugation (3000 rpm for 15 min), 1 ml of supernatant was added to 1ml of 0.5 N NaOH. A 0.2 ml sample (in a 96-well plate) was read at 560nm using Molecular Devices Spectra Max 190 spectrophotometer. The extentof gastric emptying and intestinal transit were expressed as percent oftotal phenol red recovered per rat.

Gastric emptying and intestinal transit of this semi-solid meal wereincreased by approximately two-fold following i.v. administration of0.1-1.0 μg/kg BW BV- or E. coli-produced Zven1. Inhibitory effects ofgastric emptying and intestinal transit were observed using higher doses(10-100 ug/kg BW for i.p. dosing; 30 ug/kg BW for i.v. dosing) of BV-and E. coli-Zven1. The inhibitory observations were especially evidentwhen these higher doses of Zven1 were administered i.v. at 1 minutefollowing test meal administration, or when administered i.p. at 10minutes prior to test meal administration. Similar results were observedwhen Zven2 was administered i.v. at 30 μg/kg.

Example 22 Effect of i.v. BV- and E. Coli-Produced Zven1 on GastricEmptying and Intestinal Transit of a Phenol Red Semi-Solid Meal in Mice

Female C57B1/6 mice, 8 to 10 weeks old, were used for the study, whichconsisted of eight treatment groups and ˜9 mice per group. The animalswere fasted for 20 hrs in cages containing floor screens, and allowedaccess to water. Animals were weighed to determine proper dose, andtheir average weight was used to adjust the protein concentration. Zven1protein (in stock solutions of either 20 mM Mes buffer/20 mM NaCl pH6.5; or in PBS, pH 7.2) dilutions were prepared in siliconized tubesjust prior to injections. Doses were based on the average weight of thestudy animals (approximately 20 g) and adjusted with saline 0.1% w/v BSAto 0.1 ml injection volumes per mouse. Zven1 and vehicle treatments wereadministered via i.v. tail vein injection 1-2 minutes prior to receiving0.15 ml phenol red test meal as an oral gavage. The test meal consistedof 1.5% w/v aqueous methylcellulose solution (medium viscosity carboxymethylcellulose from Sigma with a final viscosity of 400-800centipoises; catalog # C-4888; lot #108H0052) containing anon-absorbable dye, 0.05% phenol red (Sigma catalog # P-4758; lot#120K3660). Twenty minutes post-administration of the test meal, animalswere euthanized and stomach and intestinal segments removed. The smallintestine was measured and divided into three equal segments: proximal,mid and distal gut. The amount of phenol red in each sample wasdetermined by spectrophotometric analysis (as described above for inExamples 20 and 21) and expressed as the percent of total phenol redrecovered per mouse. These values were used to determine the amount ofgastric emptying and gut transit per tissue collected.

Results indicated that there were increases in gastric emptying andintestinal transit in mice treated with i.v. Zven1 at doses ˜1-10 ug/kgBW. Trends toward inhibition of gastric emptying and intestinal transitwere observed using higher doses (>50 ug/kg i.v. in mice) of Zven1.

Example 23 Effect of BV- and E. coli-Produced Zven1 on Gross Morphologyof Stomach and Intestines of Urethane-Anesthetized Rats

Studies were conducted in urethane-anesthetized male Sprague-Dawley ratsto determine whether i.v. administration of BV- or E. coli Zven1 (dosesup to and including 30 ug/kg BW; a dose known to induce intestinalmotility) affected the gross appearance of the stomach and smallintestine.

Rats were fasted (with access to water) on double floor grates in cleancages for 19 h. Between 07:00 and 08:30 am, rats received an i.pinjection of urethane (0.5 ml/100 g BW of a 25% solution) and had ajugular venous catheter inserted. Anesthetized rats were returned totheir cages and kept on warming pads (maintained at 37° C.) throughoutthe day, with additional i.p. doses of urethane administered as needed.An appropriate level of anesthesia was monitored using the toe-pinchreflex test.

At ˜5 minute intervals between animals saline was administered via thejugular vein, followed by either vehicle (PBS) or BV- or E.coli-produced Zven1 at increasing doses (3, 10 and 30 ug/kg BW; 0.1 mlinjection volume) every hour for 3 hours (total of 43 ug/kg BW). Zven1protein dilutions were prepared just prior to injection. Dose was basedon the weight of the study animal (approximately 225 grams) and adjustedso that it was contained in 0.1 ml total volume of diluent (saline/0.1%BSA). Protein was diluted using siliconized microfuge tubes. Rats alsoreceived infusions of saline via Harvard pumps at a rate of 0.5 ml perhour. Approximately 8-9 hours later following the initial dose ofurethane, rats were sacrificed by cervical dislocation (underanesthesia) and their stomachs and small intestine removed forinspection and morphological evaluation.

There was no evidence of gastric or intestinal lesions in any of therats. A vehicle-treated rat had some dark fluid within a small segmentof the intestinal lumen; there was not any dark fluid observed in theZven1-treated rats. There was a significant amount of mucous within theintestinal lumen in all treatment groups, most likely as a result of theurethane anesthesia and fasting protocol.

Example 24 Effects of BV-Produced Zven1 on In Vivo GastrointestinalContractility in Anesthetized Experimental Mammals

“Sonomicrometry” is a technique, which utilizes piezoelectric crystalsto measure gastrointestinal distensibility, compliance, and tone in vivo(Sonometrics, Corp. Ontario, Canada). Crystals can be placed anywherealong the gastrointestinal tract in experimental mammals. Peristalticand segmentation contractions in the stomach and/or intestine can thenbe accurately quantified and qualified with great detail in response tothe administration of Zven1. This system offers a great deal of detailedand sophisticated outcome measures of intestinal motility/contractility.

This method of digital ultrasonomicrometry was used to investigatemotility and/or contractility in the ileum, jejunum, cecum and proximalcolon as described by Adelson et al. Gastroenterology 122, A-554. (2002)in ten rats (two groups of 5 male Sprague-Dawley rats) following an i.v.infusion of the vehicle (saline/0.1% w/v BSA) and escalating doses ofBV-produced Zven1. For these experiments, piezoelectric crystals wereattached using a small drop of cyanoacrylate glue (Vetbond, 3M AnimalCare, St. Paul, Minn.) to the relevant intestinal locations. Afterlaparatomy the urethane anesthetized rats were maintained at 37° C. viaa feedback-controlled heater. Sonometric distance signals were acquiredcontinuously at a rate of 50 samples/sec via a digital sonomicrometer(TRX-13, Sonometrics Corp, London ONT) connected to a Pentium III classcomputer running SonoLAB software (Sonometrics Corp, London, Ontario,Canada). Digitally-acquired distance data were simultaneously recordedas analog signals via an installed 4-channel DAC. These sonometricanalog signals, along with all analog physiological data (rectaltemperature, blood pressure, EKG, respiratory rate) were acquired usinga Microl401 A/D interface (Cambridge Electronic Design, Ltd, Cambridge)connected to a Pentium II class computer running Spike 2 (CambridgeElectronic Design, Ltd, Cambridge) data acquisition software to allowreal-time observation and analysis of experiment progress. This methodallows simultaneous observation of distance measurements for 4 crystalpairs. Baseline levels were obtained between each vehicle and Zven1infusion. Both circular and longitudinal motion were monitored usingtriads of piezoelectric crystals 1 mm in diameter (Sonometrics Corp.)affixed so that two of the three were oriented parallel to thelongitudinal axis and the third was oriented to the perpendicular axis.

Motility responses to applied stimuli may comprise tonic and/or phasiccomponents. Tonic and phasic components of responses were analyzedseparately. The tonic component of the trace was obtained by replacingeach point in the trace with the median value of the trace over thesurrounding 10 s. The phasic component was obtained by applying to theoriginal trace the inverse operation of a smoothing function with a 10 swindow, i.e. by removing the ‘DC component’ with a time constant of 10s. Tonic responses were analyzed in terms of mean value during aresponse, 1-min maximum excursion from baseline, duration of response,and integrated response (mean normalized response times duration).Phasic activity was analyzed in terms of its rate and amplitude. Changesin relationships between motility in different gut regions measuredsimultaneously were analyzed using cross-correlation of continuoussignals and event correlations of peak positions.

Strong contractility responses were observed in the ileum ofZven1-treated rats at i.v. doses as low as 3 ug/kg BW; contractions werealso noted in the jejunum and duodenum, though not as strong as thoseobserved for the ileum. Responses associated with a relaxation wereobserved in the proximal colon.

Example 25 Effects of ip. Administration of BV-Produced Zven1 on DistalColonic Transit in Conscious Mice

Adult male C57/BL6 mice (6-8 weeks of age; Harlan, San Diego, Calif.)were used for this study with 6-10 mice per treatment group. Mice weremaintained on a 12:12-h light-dark cycle with controlled temperature(21-23° C.) and humidity (30-35%), and were group housed in cages withfree access to food (Purina Chow) and tap water. Mice were deprived offood for 18-20 h, with free access to water before the experiments.BV-produced Zven1 in stock solution of 20 mmol MES and 20 mmol NaCl atpH 6.5 was stored at −80° C. On the day of the experiment, Zven1 wasdiluted to 0.9% NaCl with 0.1% BSA. The pH for both vehicle and Zven1 atvarious doses was 6.5.

Distal colonic transits were measured as previously described (MartinezV, et al. J Pharmacol Exp Ther 301: 611-617(2002.)). Fasted mice hadfree access to water and pre-weighed Purina chow for a 1-h period, thenwere briefly anesthetized with enflurane (1-2 min; Ethrane-Anaquest,Madison, Wis.) and a single 2-mm glass bead was inserted into the distalcolon at 2 cm from the anus. Bead insertion was performed with a glassrod with a fire-polished end to avoid tissue damage. After beadinsertion the mice were placed individually in their home cages withoutfood and water. Mice regained consciousness within a 1-2 min period andthereafter showed normal behavior. Distal colonic transit was determinedto the nearest 0.1 min by monitoring the time required for the expulsionof the glass bead (bead latency).

At the end of the 1 h feeding period, mice were briefly anesthetizedwith enflurane for bead insertion into the colon followed by theintraperitoneal injection of either vehicle, or Zven1 (3, 10, 30, or 100μg/kg). Animals were returned to their home cages without food or waterand the bead expulsion time was monitored. Results were expressed asMean±S.E. and analyzed using one-way ANOVA.

In mice, fasted for 18-20 h, re-fed for 1 h, Zven1 injected i.p. (3, 10,30, and 100 μg/kg) showed no significant changes in bead expulsion timein response to the i.p. injection of BV-Zven1 (3, 10 and 30 μg/kg):32.7±6.1, 23.1±14.5 and 34.2±15.6 min respectively compared with21.1±13.9 min in i.p. vehicle injected group. In a second group of mice,treated similarly except administered higher doses of BV-Zven1, themeasurement of distal colonic transit showed a dose-related tendency toincrease the time at which the bead is expelled in response to the i.p.injection of BV-Zven1 (30 and 100 μg/kg) (29.8±7.8 and 35.1±3.7 minrespectively compared with 22.3±5.7 min after i.p. injection of vehicle)although changes did not reach statistical significance.

Example 26 Expression of GPR73a and GPR73b in Rat Gastrointestinal Tract

Rats were fasted overnight and sacrificed. Intestines and stomachs wereisolated and four-centimeter tissue sections from the stomach throughthe end of the colon were immediately flash frozen in liquid nitrogen.Acid-Phenol extraction method was used for RNA isolation. Briefly,tissue sections were grinded in liquid nitrogen then lysed/homogenizedin acid guanidium based lysis buffer (4M Guanidine isothyocyanate, 25 mMsodium citrate (pH 7), 0.5% sarcosyl), NaOAc (0.1M finalconcentration)+βME (1:100). Lysates were spun down; supernatants weremixed with equal volume of acid phenol and 1/10 volume chloroform. Afterspinning down, equal volume of Isopropanol was added to the aqueouslayer. Samples were incubated at −20° C. then pelleted down by spinning.Pellets were washed with 70% EtOH and then resuspended in DEPC treatedwater.

Taqman EZ RT-PCR Core Reagent Kit (Applied biosystems, Foster City,Calif.) was used to determine GPR73a and GPR73b receptor expressionlevels. Following manufacturer's instructions, a standard curve wasprepared using one of the RNA isolates which had a high quality RNA andwhich showed expression of both receptors at the same level. Standardcurve dilutions of this RNA sample were prepared at the followingconcentrations: 500 ng/μl, 250 ng/μl, 100 ng/μl and 12.5 ng/μl. Thesestandard curve dilutions were first used to test the primers designedfor GPR73a and GPR73b genes and for a housekeeping gene, rodentglyceraldehyde-3-phosphate dehydrogenase (GAPDH). Once the workingconditions of primer and standard curve were established, RNA samplesisolated from rat were tested.

The RNA samples were thawed on ice and diluted to 100 ng/μl inRNase-free water (Invitrogen, Cat #750023). Diluted samples were kept onice during the experiment. Using the TaqMan EZ RT-PCR Core Reagent Kit(Applied Biosystems, Cat# N808-0236), master mix was prepared forGPR73a, GPR73b receptors and for the house keeping gene. To assaysamples in triplicate, 3.5 μl of each RNA samples were aliquoted. Forpositive controls, 3.5 μl of each standard curve dilutions were used inplace of sample RNA. For the negative control, 3.5 μl RNase-free waterwas used for the no template control. For endogenous controls (rodentGAPDH message), 3.5 μl of both standard curve dilutions and the sampleRNAs were aliquoted. Then 84 μl of PCR master mix was added and mixedwell by pipetting.

A MicroAmp Optical 96-well Reaction Plate (Applied Biosystems Cat#N801-0560) was placed on ice and 25 μl of RNA/master mix was added intriplicates to the appropriate wells. Then MicroAmp 12-Cap Strips(Applied Biosystems Cat# N801-0534) were used to cover entire plate.Then the plate was spun for two minutes at 3000 RPM in the Qiagen Sigma4-15 centrifuge.

The samples were run on a PE-ABI 7700 (Perkin Elmer, now EG&G, Inc.Wellesley, Mass.). Sequence Detector was launched and the default wasset to Real Time PCR. Fluorochrome was set to FAM. Plate template wasset to indicate where standards and where unknown test samples were.

Expression for each sample is reported as a Ct value. The Ct value isthe point at which the fluorochrome level or RT-PCR product (a directreflection of RNA abundance) is amplified to a level, which exceeds thethreshold or background level. The lower the Ct value, the higher theexpression level, since RT-PCR of a highly expressing sample results ina greater accumulation of fluorochrome/product which crosses thethreshold sooner. A Ct value of 40 means that there was no productmeasured and should result in a mean expression value of zero. The Ct isconverted to relative expression value based on comparison to thestandard curve. For each sample tested, the amount of GPR73a, GPR73b andGAPDH expression level was determined from the appropriate standardcurve. Then these calculated expression values of GPR73a and GPR73b weredivided by the GAPDH expression value of each sample in order to obtaina normalized expression for each sample. Each normalized expressionvalue was divided by the normalized-calibrator value to get the relativeexpression levels. Using GraphPad Prism software, these normalizedvalues were converted to fractions in which the highest expression levelwas indicated as 1.

TABLE 15 Normalized values (represented in fractions) for GPR73a andGPR73b expressions in rat. GPR73a GPR73b normalized normalized Samplesvalue StDev N Samples value StDev N Forestomach 0.067 0.057 3Forestomach 0.063 0.013 3 Fundus 0.003 0.023 3 Fundus 0.106 0.033 3Antrum 0.000 0.016 3 Antrum 0.000 0.004 3 Pylorus/Antrum 0.041 0.016 3Pylorus/Antrum 0.104 0.005 3 Duodenum 0.107 0.035 3 Duodenum 0.205 0.0373 Jejunum-1 0.102 0.035 3 Jejunum-1 0.100 0.058 3 2 0.087 0.020 3 20.021 0.008 3 3 0.126 0.037 3 3 0.097 0.016 3 4 0.250 0.054 3 4 0.1500.042 3 5 0.268 0.030 3 5 0.123 0.022 3 6 0.240 0.024 3 6 0.177 0.037 37 0.339 0.039 3 7 0.173 0.031 3 8 0.329 0.107 3 8 0.129 0.031 3 9 0.3270.101 3 9 0.286 0.078 3 10  0.425 0.071 3 10  0.235 0.011 3 11  0.3790.011 3 11  0.147 0.016 3 12  0.577 0.076 3 12  0.253 0.068 3 13  0.5700.043 3 13  0.315 0.053 3 14  0.250 0.011 3 14  0.171 0.017 3 15  0.4920.027 3 15  0.397 0.034 3 16  0.989 0.089 3 16  0.494 0.048 3 17  0.9770.313 3 17  0.420 0.045 3 18  1.000 0.061 3 18  0.523 0.146 3 Ileum-10.797 0.080 3 Ileum-1 0.630 0.141 3 2 0.636 0.014 3 2 0.434 0.080 3 30.614 0.015 3 3 0.441 0.115 3 4 0.923 0.085 3 4 0.871 0.288 3 5 0.8070.142 3 5 0.739 0.017 3 6 0.755 0.080 3 6 1.000 0.246 3 Cecum 0.0880.020 3 Cecum 0.369 0.036 3 Proximal 0.171 0.060 3 Proximal 0.887 0.0213 Middle 0.088 0.051 3 Middle 0.209 0.047 3 Distal 0.047 0.019 3 Distal0.012 0.002 3

Example 27 Zven1 and Monoclonal Antibodies

Rat monoclonal antibodies are prepared by immunizing 4 femaleSprague-Dawley Rats (Charles River Laboratories, Wilmington, Mass.),with the purified recombinant protein from Example 6 or Example 7,above. The rats are each given an initial intraperitoneal (IP) injectionof 25 □g of the purified recombinant protein in Complete Freund'sAdjuvant (Pierce, Rockford, Ill.) followed by booster IP injections of10 □g of the purified recombinant protein in Incomplete Freund'sAdjuvant every two weeks. Seven days after the administration of thesecond booster injection, the animals are bled and serum is collected.

The Zven1-specific rat sera samples are characterized by ELISA using 1ug/ml of the purified recombinant protein Zven1 as the specific antibodytarget.

Splenocytes are harvested from a single high-titer rat and fused toSP2/0 (mouse) myeloma cells using PEG 1500 in a single fusion procedure(4:1 fusion ratio, splenocytes to myeloma cells, “Antibodies: ALaboratory Manual, E. Harlow and D. Lane, Cold Spring Harbor Press).Following 9 days growth post-fusion, specific antibody-producinghybridoma pools are identified by radioimmunoprecipitation (RIP) usingthe Iodine-125 labeled recombinant protein Zven1 as the specificantibody target and by ELISA using 500 ng/ml of the recombinant proteinZven1 as specific antibody target. Hybridoma pools positive in eitherassay protocol are analyzed further for their ability to block thecell-proliferative activity (“neutralization assay”) of purifiedrecombinant protein Zven1 on Baf3 cells expressing the receptor sequenceof GPR73a (SEQ ID NO:27) and/or GPR73b (SEQ ID NO:28).

Hybridoma pools yielding positive results by RIP only or RIP and the“neutralization assay” are cloned at least two times by limitingdilution.

Monoclonal antibodies purified from tissue culture media arecharacterized for their ability to block the cell-proliferative activity(“neutralization assay”) of purified recombinant Zven1 on Baf3 cellsexpressing the receptor sequences. “Neutralizing” monoclonal antibodiesare identified in this manner.

A similar procedure is followed to identify monoclonal antibodies toZven2 using the amino acid sequence in SEQ ID NO:5.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An isolated nucleic acid comprising the nucleic acid sequence asshown in SEQ ID NO:14.
 2. An expression vector comprising the followingoperably linked elements: a. a transcription promoter; b. the isolatednucleic acid of claim 1; and c. a transcription terminator.
 3. Theexpression vector of claim 2, wherein the the isolated nucleic acidfurther encodes an affinity tag.
 4. A cultured cell into which has beenintroduced the expression vector according to claim
 3. 5. A method ofproducing a polypeptide, comprising the step of culturing recombinanthost cells that comprise an expression vector, wherein the expressionvector comprises the isolated nucleic acid of claim 1, a transcriptionpromoter, and a transcription terminator, wherein the promoter isoperably linked with the nucleic acid, and wherein the nucleic acid isoperably linked with the transcription terminator, and wherein theprotein encoded by the nucleic acid is produced by the recombinant cell.